Devices containing dna encoding neurotrophic agents and related compositions and methods

ABSTRACT

Devices useful in the delivery of DNA encoding neurotrophic agents, anti-fibrotic agents, and related compositions are disclosed herein for use in the treatment of central and/or peripheral nervous system injury. Methods of making and using the disclosed devices and DNA are also described. In various embodiments, the invention also discloses compositions and devices that may further include a targeting agent, such as a polypeptide that is reactive with an FGF receptor (e.g., bFGF), or another ligand that binds to cell surface receptors on neuronal cells, or a support cell. The invention also discloses methods of promoting neuronal survival and regeneration via transfection of an axon as it grows through a device or composition of the present invention, or via transfection of a repair cell.

CROSS-REFERENCE TO RELATED APPLICATIONS

The application is a continuation-in-part of U.S. application Ser. No.09/088,419, filed Jun. 1, 1998, which is hereby incorporated byreference.

TECHNICAL FIELD

The present invention relates generally to the treatment of neuronsfollowing NS injury that may result from surgery, trauma, compression,contusion, transection or other physical injury, from vascularpharmacologic or other insults including hemorrhagic or ischemic damageor from neurodegenerative or other neurological diseases. Morespecifically, the invention relates to the preparation and use ofdevices for transferring neuronal therapeutic agents and/or DNA encodingneuronal therapeutic agents into the NS, including devices that are geneactivated matrices, to alter the function, gene expression or viabilityof neuronal cells therapeutically. The invention further relates toadministration of such devices, including administration of matricescontaining useful genes.

BACKGROUND OF THE INVENTION

Neuronal regeneration and restoration of neural connectivity withindenervated tissues may be desirable events following acute or chronicnervous system (NS) injury resulting from physical transection/trauma,contusion/compression or surgical lesion, vascular pharmacologic insultsincluding hemorrhagic or ischemic damage, or from neurodegenerative orother neurological diseases. Promotion of NS neuronal protection,neuronal survival and axon generation are well controlled processes thatmainly originate during embryonic development and may persist throughadulthood.

The stability of neuronal networks depends in part on the availabilityof a variety of specific architectural and biochemical cues in theneuronal environment that maintain neuronal projections, includingaxons. In the adult NS, the viability of neurons is maintained by thecontinuous retrograde flow of neurotrophic factors from the distalneuronal target to the neuronal cell body (perikaryon). Interruption ofneural connections by physical severance of axons disconnects neuronfrom target and threatens neuronal survival.

Because of the spatiotemporal regulation of cues, including neurotrophicfactors, essential for the maintenance of neural networks, axonalregrowth following NS injury is impaired by the absence of one or moreappropriate stimuli in the vicinity of the damaged neuron. For example,neurotrophins (NT, discussed below) may be primary determinants ofneuronal regeneration, and neurotrophin availability can be a primarylimiting factor for axonal regrowth. Damaged neurons may initially startto regenerate axons, as a response to transient and regulated increasesin the expression of neurotrophic factors, but regrowth is usuallyaborted within 14 days as intracellular stores of neurotrophin in theperikaryon are exhausted. Regrowth may also be inhibited in part by thedeposition of fibrotic scar tissue during the course of wound healing.The synthesis and release of growth factors by mesenchymal and glialcells within the fibrotic scar may create localized microenvironments,or “sinks”, having high growth factor concentrations. Becauseneurotrophin dependent axonal regeneration obligatory proceeds up aconcentration gradient of the neurotropic factor, axonal entrapmentwithin a growth factor sink may result. Following axonal injury, aneuron may be deprived of essential maintenance signals (e.g.,neurotrophic factors that ordinarily would be supplied from distaltarget regions through an intact axon), and may die. Consequently,reconnection of neural pathways is prevented and functional recovery maybe compromised.

Efforts to induce axonal regrowth following NS injury have includeddirect or indirect administration of neurotrophic compounds at or nearlesion sites. According to such approaches, a neurotrophic compound maybe directly applied at or near a lesion, or may be indirectly introducedto the damaged tissue by a transplanted cell secreting theneurotrophin(s). These methods often produce localized sinks of highneurotrophin concentration at the lesion site in which axons may becomeentrapped. Thus, axonal extension beyond the lesion and along thedamaged projection tracts may be impossible. Failure to re-establishneural connections and the ensuring neuronal atrophy may result incomplete loss of function.

Another approach designed to promote axonal regrowth after NS injuryutilizes recombinant viral vectors to deliver therapeutic genes encodingneurotrophic factors. Depending on the viral vector construct anddelivery vehicle used, such approaches may under certain circumstances,(i) elicit inappropriate antiviral immune responses, (ii) promoteundesirable viral toxic effects, (iii) have limited efficacy due, forexample, to inefficiency of genetically altered viral gene promotersequences, (iv) be tumorigenic and/or (v) lack specificity regarding thecell type to which therapeutic genes are delivered. Poor targeting ofsuch recombinant viral vectors to specific cell types, for example, maylimit the value of such an approach and may establish localizedaccumulations of therapeutic gene products at the site of vectordelivery, giving rise to the problems associated with localized growthfactor sinks and axonal entrapment.

In view of these and other problems associated with neuroregenerativetherapy, there is a compelling need for improved and more effectivetreatments that are free of the above disadvantages.

The present invention exploits the use of gene activated matrices that,when administered into a NS lesion site or along the axonal projectiontract proximal to a lesion, deliver high amounts of nucleic acidsencoding a desired neuronal therapeutic product by retrograde axonaltransport to distant, targeted neuronal cell perikaryons withoutinducing localized sinks of active product that may lead to axonalentrapment, while providing other related advantages.

SUMMARY OF THE INVENTION

The compositions and methods of the present invention may be usefulwherever neuronal regeneration and restoration of connectivity withinneural networks is sought, for example following any acute or chronic NSinjury resulting from physical transection/trauma, contusion/compressionor surgical lesion, vascular pharmacologic insults including hemorrhagicor ischemic damage, or from neurodegenerative or other neurologicaldiseases.

NS injury resulting from physical transection/trauma, vascularpharmacologic insults and/or neurological diseases may further includemechanical insult and may also include NS injury resulting from burns orother chemical exposure. Such exposure may include but need not belimited to exposure to toxic compounds such as carbon monoxide or othermetabolic poisons, or exposure to free radicals, as may also accompanyaging or contribute to the pathogenesis of neurodegenerative disease.For example, increased levels of reactive oxygen species may be present,and may correlate with sites of neurodegeneration, in diseases such asAlzheimer's disease, Parkinson's disease or Huntington's disease.

Interruption of neural connections may be a consequence of acute orchronic NS injury leading to physical severance of axons that threatensneuronal survival, as described above. Accordingly, the compositions andmethods of the present invention may delay cell degeneration and celldeath by restoring the continuous retrograde flow of neurotrophicfactors, from distal neuronal targets to neuronal perikarya, that isessential for maintenance of neural networks.

A considerable amount of work has been directed to the development ofbiocompatible matrices for use in medical implants, including thosespecifically for connective tissue implantation such as in bone or woundhealing. In context of the present invention, a matrix may be employedin association with the gene or DNA coding region encoding a neuronaltherapeutic agent in order to easily deliver the gene to the site of NSinjury. The matrix is thus a “biofiller” that provides a structure forthe regulated regeneration of neuronal axons. Such matrices may beformed from a variety of materials presently in use for implantedmedical applications.

According to the present invention, compositions and methods areprovided for matrix mediated delivery of agents, and in preferredembodiments neuronal therapeutic encoding agents, that promote neuronalregeneration and survival.

In one aspect the invention provides a device for promoting neuronalregeneration, comprising a gene activated matrix comprising abiocompatible matrix and at least one neuronal therapeutic encodingagent having an operably linked promoter. In another aspect theinvention provides a device for promoting neuronal survival, comprisinga gene activated matrix comprising a biocompatible matrix and at leastone neuronal therapeutic encoding agent having an operably linkedpromoter. In certain embodiments of these aspects, the promoter is aninducible promoter and in certain embodiments the promoter is a tissuespecific promoter. In certain embodiments the promoter is GAP43promoter, GFAP promoter, neuron specific enolase promoter, FGF-receptorpromoter, elastase I gene control region, immunoglobulin gene controlregion, alpha-1-antitrypsin gene control region, beta-globin genecontrol region, myelin basic protein gene control region, myosin lightchain 2 gene control region, RSV promoter, vaccinia virus 7.5K promoter,SV40 promoter, HSV promoter, MLP adenovirus promoter, MMTV LTR promoter,CMV promoter, metallothionein promoter, a promoter having at least onecAMP response element, tie promoter, VCAM-1 promoter, alpha V-beta 3integrin promoters, ICAM-3 promoter, CD44 promoter, CD40 promoter, notch4 promoter, or an event type-specific promoter. In other embodiments thepromoter is a neuronal cell specific promoter, which in certain furtherembodiments may be GAP43 promoter, FGF receptor promoter or neuronspecific enolase promoter.

In certain embodiments, the neuronal therapeutic encoding agent encodesa neurotrophic factor, which in certain further embodiments may be amember of the neurotrophin family and in certain other furtherembodiments may be a member of the FGF family. In certain of theseembodiments the neurotrophic factor may be nerve growth factor (NGF),brain-derived neurotrophic factor (BDNF), cardiotrophin-1 (CT-1),choline acetyltransferase development factor (CDF), ciliary neurotrophicfactor (CNTF), oncostatin M (OSM); fibroblast growth factor-1 (FGF-1),FGF-2, FGF-5, glial cell-line-derived neurotrophic factor (GDNF),insulin, insulin-like growth factor-1 (IGF-1), IGF-2, interleukin-6(IL-6), leukemia inhibitor factor (LIF), neurite promoting factor (NPF),neurotrophin-3 (NT-3), NT-4, platelet-derived growth factor (PDGF),protease nexin-1 (PN-1), S-100, transforming growth factor-1 (TGF-β), orvasoactive intestinal peptide (VIP).

In some embodiments, the neuronal therapeutic encoding agent encodes aninhibitor of an antagonist of axonal generation or regeneration, and incertain further embodiments the inhibitor of an antagonist of axonalgeneration or regeneration is an inhibitor of TGF-beta. In certainembodiments, the inhibitor of TGF-beta is decorin, a TGF-beta inhibitorychemokine, an anti-TGF-beta antibody, an antisense TGF-betaoligonucleotide, a TGF-beta gene specific ribozyme or a mutatedTGF-beta. In certain embodiments, the TGF-beta inhibitory chemokine isan ELR containing member of the CXC chemokine family. In certainembodiments, the ELR containing member of the CXC chemokine family isselected from the group consisting of interleukin-8, ENA-78, GROα, GROβand GROγ. In certain embodiments, the inhibitor of TGF-beta is decorin.In certain embodiments, the inhibitor of TGF-beta is an anti-TGF-betaantibody. In certain embodiments, the inhibitor of TGF-beta is a mutatedTGF-beta.

In some embodiments, the neuronal therapeutic encoding agent isnon-covalently associated with the gene activated matrix. In certainembodiments, the neuronal therapeutic encoding agent is adsorbed to thegene activated matrix, and in certain other embodiments the neuronaltherapeutic encoding agent is absorbed in the gene activated matrix. Incertain embodiments, the neuronal therapeutic encoding agent is capableof inducing neuronal axonal generation or regeneration.

It is another aspect of the invention to provide a device for promotingneuronal regeneration, comprising a gene activated matrix, at least onesupport cell, and at least one neuronal therapeutic encoding agenthaving an operably linked promoter. It is yet another aspect of theinvention to provide a device for promoting neuronal survival,comprising a gene activated matrix, at least one support cell, and atleast one neuronal therapeutic encoding agent having an operably linkedpromoter. In certain embodiments of either of these aspects the supportcell is a Schwann cell, and in certain other embodiments the supportcell is an oligodendrocyte. In certain embodiments the support cell isan astrocyte and in certain embodiments the support cell is a microglialcell. In certain embodiments the support cell is a fibroblast. Incertain embodiments the support cell is a macrophage. In certainembodiments the support cell is an inflammatory cell which may be amacrophage, a neutrophil, a monocyte, a granulocyte or a lymphocyte.

In certain embodiments of the invention, the neuronal therapeuticencoding agent is capable of maintaining axonal generation orregeneration. In certain embodiments the gene activated matrix is animplant for a neuronal injury site. In certain embodiments the geneactivated matrix is formed upon administration. In certain embodimentsthe gene activated matrix is administered to a neuronal injury site. Incertain embodiments the gene activated matrix is a composition selectedthat is a solution, a paste, a suspension, a powder, a semisolid, anemulsion or a gel. In certain preferred embodiments, the gene activatedmatrix is a paste. In certain embodiments the neuronal therapeuticencoding agent is a nucleic acid molecule, a vector, an antisensenucleic acid molecule or a ribozyme.

In some embodiments of the invention, the device further comprises atargeting agent, which is complexed with the neuronal therapeuticencoding agent and is capable of binding a neuronal cell surfacereceptor. In certain other embodiments, the targeting agent isconjugated to the neuronal therapeutic encoding agent and is capable ofbinding a neuronal cell surface receptor. In certain other embodiments,the targeting agent is complexed with the neuronal therapeutic encodingagent and is capable of binding a repair cell surface receptor. Incertain other embodiments, the targeting agent is conjugated to theneuronal therapeutic encoding agent and is capable of binding a repaircell surface receptor. In certain other embodiments, the targeting agentis complexed with the neuronal therapeutic encoding agent and is capableof binding extracellular matrix. In certain other embodiments, thetargeting agent is conjugated to the neuronal therapeutic encoding agentand is capable of binding extracellular matrix. In certain otherembodiments, the device further comprises a nucleic acid binding domain,wherein the nucleic acid binding domain binds to a nucleic acid sequencethat forms a portion of the neuronal therapeutic encoding agent. Incertain other embodiments, the device further comprises at least onelinker that may be a cleavable linker, a linker that provides anintracellular protein sorting peptide sequence, a linker that reducessteric hindrance, a linker that provides a nuclear translocation signalor a linker that possesses a nucleic acid condensing ability. In certainother embodiments, the device contains sub-physiologic amounts of aneuronal therapeutic agent. In certain other embodiments, the devicecontains physiologic amounts of a neuronal therapeutic agent.

In certain other embodiments of the above described aspects of theinvention, the device further comprises a conduit having a lumen. Incertain embodiments, the conduit comprises the gene activated matrix andin certain other embodiments, the lumen contains the gene activatedmatrix. In certain embodiments, the conduit comprises a bioabsorbablematerial, which in certain further embodiments may be a materialcomprising gene activated matrix, type I collagen, laminin, polyglycolicacid, glycolide trimethylene carbonate (GTMC), poly(L-lactide-co-6-caprolactone), glycoproteins, proteoglycans, heparansulfate proteoglycan, nidogen, glycosaminoglycans, fibronectin,epidermal growth factor, fibroblast growth factor, nerve growth factor,cytokines, or DNA encoding growth factors and cytokines.

In certain other embodiments, the conduit comprises a non-bioabsorbablematerial, which in certain further embodiments is be polyamide,polyimide, polyurethane, segmented polyurethane, polycarbonate orsilicone. In certain other embodiments, the non-bioabsorbable materialcomprises an etched microporous synthetic polymer surface. In certainembodiments the conduit is tubular.

Turning to another aspect of the invention, a method is provided fortransferring a neuronal therapeutic encoding agent into a neuronal cell,comprising contacting a neuronal cell with any one of the devices justdescribed to effectively transfer the neuronal therapeutic encodingagent into the neuronal cell. In one embodiment, transfer of theneuronal therapeutic encoding agent comprises retrograde axonaltransport of the neuronal therapeutic encoding agent. In anotherembodiment the method further comprises expression of the neuronaltherapeutic encoding agent at a neuronal cellular site distinct from asite of contact between the device and the neuronal cell. In anotherembodiment, the device is contacted with a neuronal cell at a neuronalinjury site. In another embodiment, the device is contacted with aneuronal cell in a manner such that axonal generation or regenerationoccurs. In a further embodiment, axonal regeneration occurs withoutaxonal entrapment. In another embodiment, the device is contacted with aneuronal cell in a manner that promotes neuronal survival. In a furtherembodiment, neuronal survival is promoted without axonal entrapment. Incertain further embodiments a neural connection is established orreestablished.

It is yet another aspect of the invention to provide a method fortransferring a neuronal therapeutic encoding agent into a repair cell,comprising contacting a repair cell with any one of the devicesdescribed above to effectively transfer the neuronal therapeuticencoding agent into the repair cell. In one embodiment, the device iscontacted with a repair cell at a neuronal injury site, and in anotherembodiment the device is contacted with a repair cell in a manner suchthat axonal generation or regeneration occurs. In certain furtherembodiments axonal generation or regeneration occurs without axonalentrapment. In another embodiment, the device is contacted with a repaircell in a manner that promotes neuronal survival. In a furtherembodiment, neuronal survival is promoted without axonal entrapment. Incertain other embodiments a neural connection is established orreestablished.

In certain embodiments of the method the device contains sub-physiologicamounts of a neuronal therapeutic agent, and in certain otherembodiments the device contains physiologic amounts of a neuronaltherapeutic agent.

In still another aspect, the invention provides a method of preparing agene activated matrix for promoting neuronal regeneration and survival,comprising contacting a neuronal therapeutic encoding agent with abiocompatible matrix such that the neuronal therapeutic encoding agentassociates non-covalently with the matrix. In one embodiment, theneuronal therapeutic encoding agent is adsorbed to the gene activatedmatrix, and in another embodiment the neuronal therapeutic encodingagent is absorbed in the gene activated matrix. In certain embodimentsthe neuronal therapeutic encoding agent is a nucleic acid molecule, avector, an antisense molecule or a ribozyme.

These and other aspects of the present invention will become evidentupon reference to the following detailed description and attacheddrawings. In addition, various references are set forth below whichdescribe in more detail certain procedures or compositions (e.g.,plasmids, etc.), and are therefore incorporated by reference in theirentireties.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts expression of axonally delivered marker protein in anoptic nerve model of CNS injury.

FIG. 2 is a schematic diagram illustrating placement of a GAM at aneuronal lesion site and retrograde axonal transport of neuronaltherapeutic encoding agent to the perikaryon.

FIG. 3 depicts the results of Western immunoblot analysis showingexpression of a neuronal therapeutic encoding agent in the lesioned ratoptic nerve in vivo neuronal repair model.

FIG. 4 depicts neuronal survival 40 days after injury in animals towhich GAMs were administered.

FIG. 5 depicts neuronal survival 100 days after injury in animals towhich GAMs were administered.

FIG. 6 illustrates target specificity of conjugates having CTb as atargeting agent.

FIG. 7 illustrates bidirectional retrograde axonal delivery andexpression of targeted condensed DNAs in a rat model system of spinalcord injury.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to an in vivo method for presentation andtransfer of DNA into mammalian repair cells for the purpose ofexpressing therapeutic agents. The method of the invention involvesimplanting or placing gene activated matrices into a nervous system (NS)injury site. An NS site may be any location in which a neuronal cell ispresent, including but not limited to central nervous system (CNS) andperipheral nervous system (PNS) and any other situs at which neuronalcells or processes thereof may reside, including neuronal axonalprojection tracts.

Direct plasmid DNA transfer from a matrix to a mammalian repair cell,through stimulation of the wound healing process, offers a number ofadvantages. First, the ease of producing and purifying DNA constructscompares favorably with traditional protein production method cost.Second, matrices can act as structural scaffolds that, in and ofthemselves, promote cell in growth and proliferation. Thus, theyfacilitate the targeting of repair cells for gene transfer. Third,direct gene transfer may be an advantageous method of drug delivery formolecules that normally undergo complex biosynthetic processing or forreceptors which must be properly positioned in the cellular membrane.These types of molecules would fail to work if exogenously delivered tocells.

As used herein, a “repair cell” is defined as any cell which may bestimulated to migrate, proliferate or alter its structural and/orfunctional activity in response to tissue injury including injury to anyNS neuron. Repair cells are a component of the wound healing response.Such cells may include neurons, astrocytes, oligodendrocytes, and otherneuroglial cells; choroid plexus cells; ependymal cells; meningealcells; Schwann cells; fibroblasts, capillary endothelial cells,capillary pericytes, and other mesenchymal cells; microglial cells andinflammatory cells including macrophages, neutrophils, moncytes,granulocytes, lymphocytes, other mononuclear inflammatory cells,segmented inflammatory cells and granulation tissue cells.

The present invention also relates to pharmaceutical compositionscomprising matrices containing DNA for use in wound healing. Thecompositions of the invention are generally comprised of a biocompatiblematrix material containing DNA encoding a therapeutic protein ofinterest.

The invention overcomes shortcomings specifically associated withcurrent recombinant protein therapies for wound healing applications.First, direct gene transfer is a rational strategy that allowstransfected cells to (a) make physiological amounts of therapeuticprotein, modified in a tissue- and context-specific manner, and (b)deliver this protein to the appropriate cell surface signaling receptorunder the appropriate circumstances. For reasons described above,exogenous delivery of such molecules is expected to be associated withsignificant dosing and delivery problems. Second, repeatedadministration, while possible, is not required with gene activatedmatrix technology: cell uptake of DNA can be controlled precisely withwell-established sustained release delivery technologies, or,alternatively, integration of transfected DNA can be associated withlong term recombinant protein expression.

1. DNA Devices

The present methods and compositions may employ a variety of differenttypes of DNA molecules. The DNA molecules may include genomic, cDNAs,single stranded DNA, double stranded DNA, triple stranded DNA,oligonucleotides and Z-DNA. DNA molecules to be used according to thecompositions and methods of the present invention include neuronaltherapeutic encoding agents. Neuronal therapeutic encoding agentsinclude any nucleic acid molecules that encode proteins to promoteneuronal growth (which includes neuronal axon generation andregeneration) and/or neuronal survival (which refers to maintenance ofneuronal viability). Neuronal therapeutic encoding agents may encodeproteins that provide neuronal growth and/or neuronal survival whenexpressed in neurons, but certain proteins that are encoded by neuronaltherapeutic encoding agents may also promote neuronal growth and/orsurvival when expressed in non-neuronal cell types. For example, variouscell types in an affected tissue may participate in fibrotic scardeposition that may, inter alia, lead to undesirable growth factor sinksand may further present impediments to_NS regeneration andreestablishment of neural networks. As another example, inneurodegenerative disease CNS injury wherein CNS microglia contribute tothe pathogenesis, neuronal therapeutic agents that are targeted to andcapable of regulating the biological activity of such microglia may beuseful. For instance, neuronal therapeutic agents that are targeted toregulate the viability, biosynthetic potential or proliferative capacityof, e.g., microglia, or neuronal therapeutic encoding agents thatdeliver genes able to regulate one or more pathogenetic gene productsof, e.g., microglia, are non-limiting illustrations of additional agentsaccording to the invention that may be useful.

The DNA molecules may code for a variety of factors that promote woundhealing including extracellular, cell surface, and intracellular RNAsand proteins. Examples of such proteins include growth factors,cytokines, therapeutic proteins, hormones and peptide fragments ofhormones, inhibitors of cytokines, peptide growth and differentiationfactors, interleukins, chemokines, interferons, colony stimulatingfactors and neurotrophic factors.

The DNA molecules may also encode blocking factors, including proteinsor non-proteins that block pathological processes, thereby allowing thenatural wound healing process to occur unimpeded. Examples of suchblocking factors include antisense molecules or ribozymes that interferewith or destroy RNA function, and DNAs that, for example, encode tissueinhibitors of enzymes that destroy tissue integrity, e.g., inhibitors ofmetalloproteinase associated with arthritis. In one such embodiment,matrix metalloproteinase (MMP) expression may be helpful to nerveregeneration, e.g., by removing extracellular matrix components of thescar that block the path of axons. MMP-2 and MMP-9 are both expressed byregenerating axons (Yong et al., (1998) Trends In Neurol. Sci.21:75-78). In a preferred embodiment, DNA encoding antagonists ofcytokines or growth factors, for example, antagonists of transforminggrowth factory (TGF-β) or connective tissue growth factor (CTGF), mayact to block matrix deposition of the scar and thus promote nerveregeneration.

One may obtain the DNA segment encoding the protein or non-protein ofinterest using a variety of molecular biological techniques, generallyknown to those skilled in the art. For example, cDNA or genomiclibraries may be screened using primers or probes with sequences basedon the known nucleotide sequences. Polymerase chain reaction (PCR) mayalso be used to generate the DNA fragment encoding the protein ofinterest. Alternatively, the DNA fragment may be obtained from acommercial source.

Nucleic acid sequences that vary from those described in the literatureare also encompassed by the invention, so long as the altered ormodified nucleic acid still encodes a protein or non-protein thatfunctions to stimulate neuronal axon regeneration in any direct orindirect manner, including but not limited to effects on axonalregrowth, on neuronal survival, on the activities of other cell types inthe vicinity of a NS lesion or on wound healing generally. Thesesequences include those caused by point mutations, those due to thedegeneracies of the genetic code or naturally occurring allelicvariants, and further modifications that have been introduced by geneticengineering, i.e., by the hand of man.

Techniques for introducing changes in nucleotide sequences that aredesigned to alter the functional properties of the encoded proteins orpolypeptides are well known in the art. Such modifications include thedeletion, insertion or substitution of bases which result in changes inthe amino acid sequence. Changes may be made to increase the activity ofan encoded protein, to increase its biological stability or half-life,to change its glycosylation pattern, confer temperature sensitivity orto alter the expression pattern of the protein and the like. All suchmodifications to the nucleotide sequences are encompassed by thisinvention.

Modification of DNA may be performed by a variety of methods, includingsite-specific or site-directed mutagenesis of DNA encoding the proteinand the use of DNA amplification methods using primers to introduce andamplify alterations in the DNA template, such as PCR splicing by overlapextension (SOE). Site-directed mutagenesis is typically effected using aphage vector that has single- and double-stranded forms, such as M13phage vectors, which are well-known and commercially available. Othersuitable vectors that contain a single-stranded phage origin ofreplication may be used (see, e.g., Veira et al., Meth. Enzymol. 15:3,1987). In general, site-directed mutagenesis is performed by preparing asingle-stranded vector that encodes the protein of interest (e.g., amember of the FGF family or a neurotrophin). An oligonucleotide primerthat contains the desired mutation within a region of homology to theDNA in the single-stranded vector is annealed to the vector followed byaddition of a DNA polymerase, such as E. coli DNA polymerase I (Klenowfragment), which uses the double stranded region as a primer to producea heteroduplex in which one strand encodes the altered sequence and theother the original sequence. The heteroduplex is introduced intoappropriate bacterial cells and clones that include the desired mutationare selected. The resulting altered DNA molecules may be expressedrecombinantly in appropriate host cells to produce the modified protein.

Conservative substitutions of amino acids are well-known and may be madegenerally without altering the biological activity of the resultingmolecule. For example, such substitutions are generally made byinterchanging within the groups of polar residues, charged residues,hydrophobic residues, small residues, and the like. If necessary, suchsubstitutions may be determined empirically merely by testing theresulting modified protein for the ability to bind to and internalizeupon binding to the appropriate receptors. Those that retain thisability are suitable for use in the constructs and methods herein.

For example, certain amino acids may be substituted for other aminoacids in a protein structure without appreciable loss of interactivebinding capacity with structures such as, for example, antigen-bindingregions of antibodies or binding sites on substrate molecules. Since itis the interactive capacity and nature of a protein that defines thatprotein's biological functional activity, certain amino acid sequencesubstitutions can be made in a protein sequence, and, of course, itsunderlying DNA coding sequence, and nevertheless obtain a protein withlike properties. It is thus contemplated by the inventors that variouschanges may be made in the DNA sequences of neuronal therapeutic geneswithout appreciable loss of their biological utility or activity.

In making such changes, the hydropathic index of amino acids may beconsidered. The importance of the hydropathic amino acid index inconferring interactive biologic function on a protein is generallyunderstood in the art (Kyte and Doolittle, 1982, incorporate herein byreference). It is accepted that the relative hydropathic character ofthe amino acid contributes to the secondary structure of the resultantprotein, which in turn defines the interaction of the protein with othermolecules, for example, enzymes, substrates, receptors, DNA, antibodies,antigens, and the like.

Each amino acid has been assigned a hydropathic index on the basis ofits hydrophobicity and charge characteristics (Kyte and Doolittle,1982); these are: isoleucine (+4.5); valine (+4.2); leucine (+3.8);phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9);alanine (+1.8); glycine (−0.4); threonine (−0.7); serine (−0.8);tryptophan (−0.9); tyrosine (−1.3); proline (−1.6); histidine (−3.2);glutamate (−3.5); glutamine (−3.5); aspartate (−3.5); asparagine (−3.5);lysine (−3.9); and arginine (−4.5).

It is known in the art that certain amino acids may be substituted byother amino acids having a similar hydropathic index or score and stillresult in a protein with similar biological activity, i.e., still obtaina biological functionally equivalent protein. In making such changes,the substitution of amino acids whose hydropathic indices are within +2is preferred, those which are within +1 are particularly preferred, andthose within +0.5 are even more particularly preferred.

It is also understood in the art that the substitution of like aminoacids can be made effectively on the basis of hydrophilicity. U.S. Pat.No. 4,554,101, incorporated herein by reference, states that thegreatest local average hydrophilicity of a protein, as governed by thehydrophilicity of its adjacent amino acids, correlates with a biologicalproperty of the protein.

As detailed in U.S. Pat. No. 4,554,101, the following hydrophilicityvalues have been assigned to amino acid residues: arginine (+3.0);lysine (+3.0); aspartate (+3.0±1); glutamate (+3.0±1); serine (+0.3);asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (−0.4);proline (−0.5±1); alanine (−0.5); histidine *−0.5); cysteine (−1.0);methionine (−1.3); valine (−1.5); leucine (−1.8); isoleucine (−1.8);tyrosine (−2.3); phenylalanine (−2.5); tryptophan (−3.4).

It is understood that an amino acid can be substituted for anotherhaving a similar hydrophilicity value and still obtain a biologicallyequivalent, and in particular, an immunologically equivalent protein. Insuch changes, the substitution of amino acids whose hydrophilicityvalues are within ±2 is preferred, those which are within ±1 areparticularly preferred, and those within ±0.5 are even more particularlypreferred.

As outlined above, amino acid substitutions are generally thereforebased on the relative similarity of the amino acid side-chainsubstituents, for example, their hydrophobicity, hydrophilicity, charge,size, and the like. Exemplary substitutions which take various of theforegoing characteristics into consideration are well known to those ofskill in the art and include: arginine and lysine; glutamate andaspartate; serine and threonine; glutamine and asparagine; and valine,leucine and isoleucine.

The DNA encoding the translational or transcriptional products ofinterest, for example a neuronal therapeutic encoding agent, may berecombinantly engineered into a variety of vector systems that providefor replication of the DNA in large scale for the preparation of geneactivated matrices. These vectors can be designed to contain thenecessary elements for directing the transcription and/or translation ofthe DNA sequence taken up by neurons or by repair cells at a wound sitein vivo, such as injured neurons at an NS lesion site.

Vectors that may be used include but are not limited to those derivedfrom recombinant bacteriophage DNA, plasmid DNA or cosmid DNA. Forexample, plasmid vectors such as pBR322, pUC19/18, pUC118, 119 and theM13 mp series of vectors may be used. Bacteriophage vectors may includeλgt10, λgt11, λgt18-23, λZAP/R and the EMBL series of bacteriophagevectors. Cosmid vectors that may be utilized include, but are notlimited to, pJB8, pCV 103, pCV 107, pCV 108, pTM, pMCS, pNNL, pHSG274,COS202, COS203, pWE15, pWE16 and the charomid 9 series of vectors.Vectors that allow for the in vitro transcription of RNA, such as SP6vectors, may also be used to produce large quantities of RNA that may beincorporated into matrices. Alternatively, recombinant virus vectors maybe engineered, including but not limited to those derived from virusessuch as herpes virus, retroviruses, vaccinia virus, poxviruses,adenoviruses, adeno-associated viruses or bovine papilloma virus. Whileintegrating vectors may be used, non-integrating systems, which do nottransmit the gene product to daughter cells for many generations arepreferred for wound healing, such as neuronal axon regeneration. In thisway, the gene product is expressed during the wound healing/neuronalrepair/axonal regeneration process, and as the gene is diluted out inprogeny generations, the amount of expressed gene product is diminished.As described above, restoration of neural networks reestablishesretrograde flow of neurotrophic factors, thus obviating in certainsituations the need for constitutive expression of a GAM deliveredneuronal therapeutic encoding agent.

Methods which are well known to those skilled in the art can be used toconstruct expression vectors containing the protein coding sequenceoperatively associated with appropriate transcriptional/translationalcontrol signals. These methods include in vitro recombinant DNAtechniques, and synthetic techniques. See, for example, the techniquesdescribed in Sambrook, et al., 1992, Molecular Cloning, A LaboratoryManual, Cold Spring Harbor Laboratory, N.Y. and Ausubel et al., 1989,Current Protocols in Molecular Biology, Greene Publishing Associates &Wiley Interscience, New York.

The genes encoding the proteins of interest may be operativelyassociated with a variety of different promoter/enhance elements, whichmay include but need not be limited to promoter, enhancer, transcriptionfactor binding site and other gene expression regulatory sequences. Theexpression elements of these vectors may vary in their strength andspecifities. Depending on the host/vector system utilized, any one of anumber of suitable transcription and translation elements may be used.The promoter may be in the form of the promoter which is naturallyassociated with the gene of interest. Alternatively, the DNA may bepositioned under the control of a recombinant or heterologous promoter,i.e., a promoter that is not normally associated with that gene. In anyevent, the promoter is included as an “operably linked” promoter, whichrefers to the situation of a promoter in any embodiment of a neuronaltherapeutic encoding agent according to the present invention in such amanner as to influence the expression of the neuronal therapeutic agentencoded by the neuronal therapeutic encoding agent. For example, tissuespecific promoter/enhancer elements, including distinct promoter andenhancer sequences that are derived from different sources andengineered to produce a recombinant promoter/enhancer element, may beused to regulate the expression of the transferred DNA in specific celltypes. Examples of described transcriptional control regions exhibitingtissue specificity that may be used include but are not limited to glialfibrillary acid protein (GFAP) gene control region, which is active inastrocytes (Brenner and Messing, 1996, Methods: A Companion to Methodsin Enzymology 10:351-364); GAP43 gene control region (de Groen et al.,1995, J. Mol. Neurosci. 6:109-119); elastase I gene control region(Swift et al., 1984, Cell 38:639-646; Omitz et al., 1986, Cold SpringHarbor Symp. Quant. Biol. 50:399-409; MacDonald, 1987, Hepatology 7:42S-51S); immunoglobulin gene control region, which is active in lymphoidcells (Grosechedl et al., 1984, Cell 38:647-658; Adama et al., 1985,Nature 318:533-538; Alexander et al., 1987, Mol. Cell. Biol.7:1436-1444); FGF-receptor promoter; alpha-1-antitrypsin gene controlregion, which is active in liver (Kelsey et al., 1987, Genes and Devel.1:161-171); beta-globin gene control region, which is active in myeloidcells (Magram et al., 1985, Nature 315:338-340; Kollias et al., 1986,Cell 46:89-94); myelin basic protein gene control region, which isactive in oligodendrocyte cells in the brain (Readhead et al., 1987,Cell 48:703-712); and myosin light chain 2 gene control region, which isactive in skeletal muscle (Shani, 1985, Nature 314:283-286). Promotersisolated from the genome of viruses that grow in mammalian cells, (e.g.,RSV, vaccinia virus 7.5K, SV40, HSV, adenoviruses MLP, MMTV LTR and CMVpromoters) may be used, as well as promoters produced by recombinant DNAor synthetic techniques.

In some instances, the promoter elements may be constitutive orinducible promoters and can be used under the appropriate conditions todirect high level or regulated expression of the gene of interest.Expression of genes under the control of constitutive promoters does notrequire the presence of a specific substrate to induce gene expressionand will occur under all conditions of cell growth. In contrast,expression of genes controlled by inducible promoters is responsive tothe presence or absence of an inducing agent.

Specific initiation signals are also required for sufficient translationof inserted protein coding sequences. These signals include the ATGinitiation codon and adjacent sequences. In cases where the entirecoding sequence, including the initiation codon and adjacent sequencesare inserted into the appropriate expression vectors, no additionaltranslational control signals may be needed. However, in cases whereonly a portion of the coding sequence is inserted, exogenoustranslational control signals, including the ATG initiation codon mustbe provided. Furthermore, the initiation codon must be in phase with thereading frame of the protein coding sequences to ensure translation ofthe entire insert. These exogenous translational control signals andinitiation codons can be of a variety of origins, both natural andsynthetic. The efficiency and control of expression may be enhanced bythe inclusion of transcription attenuation sequences, enhance elements,etc.

In addition to DNA sequences encoding therapeutic proteins of interest,the scope of the present invention includes the use of ribozymes orantisense DNA molecules that may be transferred into the mammalianrepair cells. Such ribozymes and antisense molecules may be used toinhibit the translation of RNA encoding proteins of genes that inhibit adisease process or the wound healing process thereby allowing tissuerepair to take place.

The expression of antisense RNA molecules will act to directly block thetranslation of mRNA by binding to targeted mRNA and preventing proteintranslation. The expression of ribozymes, which are enzymatic RNAmolecules capable of catalyzing the specific cleavage of RNA may also beused to block protein translation. The mechanism of ribozyme actioninvolves sequence specific hybridization of the ribozyme molecule tocomplementary target RNA, followed by a endonucleolytic cleavage. Withinthe scope of the invention are engineered hammerhead motif ribozymemolecules that specifically and efficiently catalyze endonucleolyticcleavage of RNA sequences. RNA molecules may be generated bytranscription of DNA sequences encoding the RNA molecule.

It is also within the scope of the invention that multiple genes,combined on a single genetic construct under control of one or morepromoters, or prepared as separate constructs of the same or differenttypes may be used. Thus, an almost endless combination of differentgenes and genetic constructs may be employed. Certain gene combinationsmay be designed to, or their use may otherwise result in, achievingsynergistic effects on cell stimulation and regeneration, any and allsuch combinations are intended to fall within the scope of the presentinvention. Indeed, many synergistic effects have been described in thescientific literature, so that one of ordinary skill in the art wouldreadily be able to identify likely synergistic gene combinations, oreven gene-protein combinations. For example, in one embodiment,expression of genes encoding neuronal therapeutic agents combined withexpression of genes encoding anti-fibrotic or anti-inflammatorycytokines provide synergistic stimulation of neuron growth.

The term “gene” is used for simplicity to refer to a functional proteinor peptide encoding unit. As will be understood by those in the art,this functional term includes both genomic sequences and cDNA sequences.“Isolated substantially away from other coding sequences” means that thegene of interest, in this case, a therapeutic and in particular aneuronal therapeutic gene, forms the significant part of the codingregion of the DNA segment, and that the DNA segment does not containlarge portions of naturally-occurring coding DNA, such as largechromosomal fragments or other functional genes or cDNA coding regions.Of course, this refers to the DNA segment as originally isolated, anddoes not exclude genes or coding regions, such as sequences encodingleader peptides or targeting sequences, later added to the segment bythe hand of man.

This invention provides novel ways in which to utilize various knownneuronal therapeutic DNA segments and recombinant vectors. As describedabove, many such vectors are readily available, one particular detailedexample of a suitable vector for expression in mammalian cells is thatdescribed in U.S. Pat. No. 5,168,050, incorporated herein by reference.However, there is no requirement that a highly purified vector be used,so long as the coding segment employed encodes a neuronal therapeuticprotein and does not include any coding or regulatory sequences thatwould have a significant adverse effect on neurons. Therefore, it willalso be understood that useful nucleic acid sequences may includeadditional residues, such as additional non-coding sequences flankingeither of the 5′ or 3′ portions of the coding region or may includevarious internal sequences, i.e., introns, which are known to occurwithin genes.

After identifying an appropriate neuronal therapeutic encoding agent,such as a suitable gene or nucleic acid molecule, it may be insertedinto any one of the many vectors currently known in the art, so that itwill direct the expression and production of the neuronal therapeuticprotein when incorporated into a neuron. In a recombinant expressionvector, the coding portion of the DNA segment is positioned under thecontrol of a promoter. The promoter may be in the form of the promoterwhich is naturally associated with a neuronal therapeutic agent encodinggene, as may be obtained by isolating the 5′ non-coding sequenceslocated upstream of the coding segment or exon, for example, usingrecombinant cloning and/or PCR technology, in connection with thecompositions disclosed herein.

In other embodiments, it is contemplated that certain advantages will begained by positioning the coding DNA segment under the control of arecombinant, or heterologous, promoter. As used herein, a recombinant orheterologous promoter is intended to refer to a promoter that is notnormally associated with a neuronal therapeutic agent encoding gene inits natural environment. Such promoters may include those normallyassociated with other neuronal therapeutic genes, and/or promotersisolated from any other bacterial, viral, eukaryotic, or mammalian cell.Naturally, it will be important to employ a promoter that effectivelydirects the expression of the DNA segment in target cells, in neurons,for example, a GAP43, FGF-receptor or neuron specific enolase promoterand control region.

The use of recombinant promoters and/or enhancers to achieve proteinexpression is generally known to those of skill in the art of molecularbiology, for example, see Sambrook et al., (1989). The promotersemployed may be constitutive, or inducible, and can be used under theappropriate conditions to direct high level or regulated expression ofthe introduced DNA segment. The currently preferred promoters are thosesuch as GFAP promoter, GAP43 promoter, CMV, RSV LTR, the SV40 promoteralone, and the SV40 promoter in combination with various enhancerelements. For example, neuronal therapeutic agents may be targeted toneurons with the GAP43 promoter or the neuron specific enolase promoter,and anti-fibrotic agents may be targeted to astrocytes with the GFAPpromoter.

Neuronal therapeutic agent encoding genes and DNA segments may also bein the form of a DNA insert which is located within the genome of arecombinant virus, for example, a recombinant adenovirus,adeno-associated virus (AAV), herpes virus, pox virus or retrovirus. Insuch embodiments, to place the gene in contact with a neuron, one wouldprepare the recombinant viral particles, the genome of which includesthe neuronal therapeutic encoding gene insert, and simply contact the NSregion containing injured neuronal cells with the a delivery devicecontaining the virus, whereby the virus infects the cells and transfersthe genetic material.

In certain preferred embodiments, one would impregnate a matrix orimplant material with virus by soaking the material in recombinant virusstock solution, e.g., for 1-2 hours, and then contact the damaged NScells or tissues with the resultant, impregnated matrix. Cells thenpenetrate, or grow into, the matrix, thereby contacting the virus andallowing viral infection which leads to the cells taking up the desiredgene or cDNA and expressing the encoded protein.

In other preferred embodiments, one would form a matrix-nucleic acidadmixture, whether using naked DNA, a plasmid or a viral vector, bymixing the nucleic acid or construct with matrix or implant materialthat is in solution, suspension, paste, colloid or other liquid form asdescribed herein, permitting the admixture to undergo polymerization,gelation, semisolidification or the like as may be a property of theparticular material selected, and contacting the injured NS neuronalcells or tissues with the resultant admixed matrix. The matrix may thendeliver the nucleic acid into the cells following disassociation at thecell surface, or in the immediate cellular environment. Equally, thematrix admixture itself, especially a particle- or fiber-DNA admixture,may be taken up by cells to provide subsequent intracellular release ofthe genetic material. The matrix may then be extruded from the cell,catabolized by the cell, or even stored within the cell. The molecularmechanism by which a neuron-compatible matrix achieves transfer of DNAto a cell is immaterial to the practice of the present invention.

a. Gene Activated Matrix (GAM) Delivery Systems

As noted above, the complexes and the constructs of the presentinvention may be formulated into a gene activated matrix (GAM) foradministration at a NS lesion site. This embodiment of the presentinvention relates to an in vivo method for presentation and transfer ofDNA into host neurons and/or repair cells for the purpose of expressingtherapeutic agents. Gene activated matrices are disclosed in U.S. Pat.No. 5,763,416 and in published PCT App. No. WO 97/38729, the disclosuresof which are hereby incorporated by reference in their entirety.

As also noted above, neurons in the NS may react to physical injury bytransiently sprouting regenerating axons that may respond to neuronaltherapeutic factors and/or other trophic spatiotemporal signals,provided such signals are delivered before the onset of neuronal atrophyand necrosis. Successful neuronal regeneration and functionalrestoration of NS neural networks after traumatic injury may becompromised by tissue repair mechanisms of non-neural cell types. Forexample, as discussed above, inappropriate scar tissue deposition mayobstruct projection tracts for neuronal regeneration by creating sinksof neurotrophic factors that lead to axonal development, and glial scarmatrix may physicochemically inhibit axonal regeneration. In mosttissues wound healing is usually a coordinated, stereotyped sequence ofevents that includes (a) tissue disruption and loss of normal tissuearchitecture; (b) cell necrosis and hemorrhage; hemostasis (clotformation); (c) infiltration of segmented and mononuclear inflammatorycells, with vascular congestion and tissue edema; (d) dissolution of theclot as well as damaged cells and tissues by mononuclear cells(macrophages) (e) formation of granulation tissue (fibroplasia andangiogenesis). This sequence of cellular events has been observed inwounds from all tissues and organs generated in a large number ofmammalian species (See Berry et al., 1998 In: CNS Injuries: Responsesand Pharmacological Strategies, A. Logan and M. Berry, eds., CRC Press,Boca Raton, Fla.; Gailet et al., 1994, Curr. Opin. Cell. Biol.6:717-725). Therefore, the cellular sequence described above is auniversal aspect of the repair of all mammalian tissues.

The invention is based on the discovery that many types of repair cellsinvolved in the wound healing process, including by way of illustrationand not limitation astrocytes, glial cells, microglial cells andfibroblasts, will naturally proliferate and migrate to the site oftissue injury and infiltrate the gene activated matrix (GAM). In thecase of (generally non-proliferating) neuronal cells attempting axonalregeneration following injury, the axonal sprouts may similarly contactand/or invade the GAM. Surprisingly, these repair cells, which arenormally difficult to efficiently transfect either in vitro or in vivo,are extremely efficient at taking up and expressing DNA when activatedby the wound healing process. Regenerating neuronal axons may alsocontact and/or invade the GAM, providing an opportunity for DNA uptakeand delivery to perikarya by retrograde transport. Thus, the methods ofthe present invention are designed to efficiently transfer DNA moleculesencoding therapeutic agents to regenerating neurons and/or repair cells.The devices and methods involve the administration, within a host at thesite of NS injury, of GAM containing constructs, complexes or conjugatesincluding a biocompatible matrix and a neuronal therapeutic encodingagent and, optionally, one or more of a ligand, a promoter, a nucleicacid binding domain, a linker, translational products (i.e., therapeuticproteins), transcriptional products (i.e., antisense nucleic acids orribozymes) or any other agent that may be a neuronal therapeutic agent.

For example, as the regenerating axon may grow into and contact the GAM,it may take up DNA encoding a therapeutic agent, which DNA is deliveredto the perikaryon by retrograde axonal transport. The transfectedneurons may thereby serve as distal bioreactors producing therapeuticagents that influence the local repair environment through expression ofthe delivered DNA. As described above, expression of neuronaltherapeutic agents may in certain embodiments of the invention furthercomprise regulated transcription of a neuronal therapeutic agent, forexample through a neuron specific promoter and/or a nucleic acid bindingdomain. For example, neurotrophic factors, growth factors, cytokines orother neuronal therapeutic agents produced by the transfected neuronsmay stimulate and amplify the cascade of physiological events normallyassociated with the neuronal regeneration process. Because the GAM mayinclude DNA encoding a neuronal therapeutic agent but not thetherapeutic agent itself, elaboration of a therapeutic agent sink withinthe GAM loaded lesion and resulting axonal entrapment may be avoided,particularly if the therapeutic agent is engineered to be retainedintracellularly and to exert its neuronal therapeutic effectintracellularly, or where the therapeutic agent is biosyntheticallyproduced and released extracellularly at sufficiently low levels toavoid a sink effect.

Alternatively, the regenerating axons of neurons, or other involved celltypes, may take up and express DNA encoding proteins that inhibit theactivity of antagonists of the neuronal survival/axonalgeneration/regeneration process. Such antagonists may operate on anycell type in the vicinity of an NS lesion and by any mechanism, director indirect, to interfere with NS wound healing. Accordingly, in certainembodiments of the invention a neuronal therapeutic encoding agent mayencode an inhibitor of such an antagonist of axonal generation orregeneration. As a non-limiting example, for instance, anti-scarringactivity of the cell surface proteoglycan decorin is related toretention by decorin of TGF-β, thereby preventing binding of TGF-β toits receptor. (Border et al., Nature 360:361, 1992; Hausser et al., FEBSLett. 353:243-245, 1994.) Accordingly, delivery and expression ofdecorin-encoding genes at NS injury sites, as provided by the presentinvention, may similarly discourage local scarring while favoring axonalregeneration. The DNA may also encode antisense or ribozyme RNAmolecules that may be used to inhibit translation in neural ornon-neural cells of mRNAs encoding inflammatory proteins, scar tissuecomponents or other factors that inhibit neural regeneration. As anothernon-limiting example, extracellular matrix deposition accompanying scarformation and that is promoted by TGF-beta may be impaired by a neuronaltherapeutic encoding agent encoding a TGF-beta inhibitory chemokine, forexample the ELR containing members of the CXC family of chemokinesdescribed by Moore et al. (1998 J. Invest. Med. 46:113). As anothernon-limiting example, anti-TGF-beta antibodies may be useful neuronaltherapeutic agents because of their ability to interfere with TGF-betamediated scar tissue generation. (See, e.g., Gharaee-Kermani et al.,1996 J. Biol. Chem. 271:17779.)

The gene activated matrices of the invention can be transferred to thepatient using a variety of techniques. For example, when stimulatingneural regeneration, the matrices are transferred directly to the siteof the NS lesion, e.g., the axotomized neuron. Since the method of theinvention is based on natural axonal sprouting in response to axonotomyleading to axonal entry into the gene activated matrix located at thelesion site, and the consequential uptake of e.g., DNA, it is understoodthat the matrices must be transferred into a site in the body where NSlesions and axonal sprouting have been induced.

One particularly important feature of the present invention is that therepair process may be engineered to result in either neural regenerationand/or the formation of scar tissue. Around a suture, for example, itmay be desirable to form scar tissue to hold inherently weak tissuetogether. At the site of the actual NS injury (e.g., the neuronallesion), however, the expression of neuronal therapeutic agents mayresult in regeneration of neurons without the formation of scar tissue.In many instances, such neuronal regeneration is desirable. As describedabove, overexpression of neuronal therapeutic agents at NS lesion sitesmay lead to therapeutic agent sinks and resulting axonal entrapment. Itis therefore within the scope of this aspect of the invention to providenucleic acid constructs, for use as neuronal therapeutic agent encodingmolecules in GAMs, that may qualitatively or quantitatively regulate thebiosynthesis and localization of neuronal therapeutic agents in a mannerthat avoids formation of such sinks. For example, by way of illustrationand not limitation, pharmacologically inactive genetic constructsencoding polypeptide domains that direct a neuronal therapeutic agent toa particular subcellular localization, or constructs having promotersthat permit only restricted expression levels of such agents, maycircumvent the generation of therapeutic agent sinks. Accordingly, whenthe delivered agent is a neuronal therapeutic encoding agent that ispharmacologically inactive at the lesion site where it is administered,the problem of axonal entrapment in lesion associated therapeutic agentsinks is overcome by axonal transport of the therapeutic encoding agentaway from the lesion prior to biosynthesis of the encoded therapeuticagent at a location distinct from the lesion site. These and other meansfor regulating neuronal therapeutic agent encoding gene expression arewithin the scope of the present invention. Therefore, the methods ofinvention may be used to stimulate NS tissue repair and/or woundhealing, either with or without the formation of scar tissue, dependingon the type and amount of therapeutic agent expressed.

b. The Gene Activated Matrix

Any biocompatible matrix material containing DNA encoding a therapeuticagent of interest, e.g., therapeutic proteins, or transcriptionalproduct, e.g., antisense or ribozymes, can be formulated and used inaccordance with the invention. Further information regarding useful GAMmaterials may be found in the disclosure of U.S. Pat. No. 5,763,416, forexample, which is incorporated by reference herein.

The gene activated matrices of the invention may be derived from anybiocompatible material. Such materials may include, but are not limitedto, biodegradable or nonbiodegradable materials formulated intoscaffolds that support cell attachment and growth, powders or gels.Matrices may be derived from synthetic polymers or naturally occurringproteins such as collagen, fibrin or other extracellular matrixproteins, or other structural macromolecules.

The DNA incorporated into the matrix may encode any of a variety oftherapeutic proteins depending on the envisioned therapeutic use. Suchproteins may include neuronal therapeutic agents such as neurotrophins,growth factors, cytokines, enzymes, hormones, proto-oncogenes or anyother proteins capable of regulating the growth, differentiation orphysiological function of neurons and/or other cells at or near NSlesions. The DNA may also encode antisense or ribozyme molecules thatblock the translation of proteins that promote scar formation, thatinhibit wound repair and/or that induce inflammation. As describedabove, the DNA may also encode antagonists of cytokines or growthfactors, which cytokines or growth factors promote extracellular matrixdeposition and scar formation. Thus, for example, antagonists of TGF-βor CTGF may promote nerve regeneration.

The transferred DNA need not be integrated into the genome of the targetcell; indeed, the use of non-integrating DNA in the gene activatedmatrix is a preferred embodiment of the present invention. In this way,when the neural network pathway is restored and the gene product is nolonger needed, the gene product may no longer be expressed.

Therapeutic kits containing a biocompatible matrix and DNA form anotheraspect of the invention. In some instances the kits will containpreformed gene activated matrices thereby allowing the physician todirectly administer the matrix within the body. Alternatively, the kitsmay contain the components necessary for formation of a gene activatedmatrix. In such cases the physician may combine the components to formthe gene activated matrices which may then be used therapeutically byplacement within the body. In one embodiment of the invention thematrices may be used to coat surgical devices such as suture materialsor implants. In yet another embodiment of the invention, gene activatedmatrices may include ready to use sponges, tubes, band-aids, lyophilizedcomponents, gels, patches or powders and telfa pads, to name a fewexamples.

c. The Matrix Materials

In one aspect of the invention, compositions are prepared in which theDNA encoding the therapeutic agent of interest (e.g., a neuronaltherapeutic agent) is associated with or impregnated within a matrix toform a gene activated matrix. The matrix compositions function (i) tofacilitate in growth of regenerating axons (targeting); and (ii) toharbor DNA (delivery). Once the gene activated matrix is prepared it isstored for future use or placed immediately at or near the site of thewound.

The type of matrix that may be used in the compositions, devices andmethods of the invention is virtually limitless and may include bothbiological and synthetic matrices. The matrix will have all the featurescommonly associated with being “biocompatible”, in that it is in a formthat does not produce an adverse, allergic or other untoward reactionwhen administered to a mammalian host. Such matrices may be formed fromeither natural or synthetic materials, or both. The matrices may benon-biodegradable in instances where it is desirable to leave permanentstructures in the body; or biodegradable where the expression of thetherapeutic protein is required only for a short duration of time. Forexample, the matrices may take the form of sponges, implants, tubes,telfa pads, band-aids, bandages, pads, lyophilized components, gels,patches, powders or nanoparticles. In addition, matrices can be designedto allow for sustained release of the DNA over prolonged periods oftime. Such sustained release of a therapeutic DNA construct, andcorresponding sustained expression of neuronal therapeutic agentsencoded thereby, may be preferred in situations where long neural tractregrowth is sought, for example, in spinal cord or optic system repair.

The choice of matrix material will differ according to the particularcircumstance and the site of the lesion that is to be treated. Matricessuch as those described in U.S. Pat. No. 5,270,300 or 5,763,416,incorporated herein by reference, may be employed. Physical and chemicalcharacteristics, such as, e.g., biocompatibility, biodegradability,strength, rigidity, interface properties and even cosmetic appearancemay be considered in choosing a matrix, as is well known to those ofskill in the art. Appropriate matrices will both deliver the DNAmolecule and also act as an in situ scaffolding through whichregenerating axons may migrate.

Where the matrices are to be maintained for extended periods of time,non-biodegradable matrices may be employed, such as sinteredhydroxyapatite, bioglass, aluminates, other bioceramic materials andmetal materials, particularly titanium. A suitable ceramic deliverysystem is that described in U.S. Pat. No. 4,596,574, incorporated hereinby reference. The bioceramics may be altered in composition, such as incalcium-aluminate-phosphate; and they may be processed to modifyparticular physical and chemical characteristics, such as pore size,particle size, particle shape, and biodegradability. Polymeric matricesmay also be employed, including acrylic ester polymers and lactic acidpolymers, as disclosed in U.S. Pat. Nos. 4,521,909, and 4,563,489,respectively, each incorporated herein by reference. Particular examplesof useful polymers are those of orthoesters, anhydrides,propylene-cofumarates, or a polymer of one or more γ-hydroxy carboxylicacid monomers, e.g., γ-hydroxy auric acid (glycolic acid) and/orγ-hydroxy propionic acid (lactic acid).

The constructs and complexes may be prepared with carriers that protectthem against rapid elimination from the body, such as time releaseformulations or coatings. Such carriers include controlled releaseformulations, such as, but not limited to, implants andmicroencapsulated delivery systems, and biodegradable, biocompatiblepolymers, such as ethylene vinyl acetate, polyanhydrides, polyglycolicacid, polyorthoesters, polylactic acid and others. For example, thecomposition may be applied during surgery using a sponge, such as acommercially available surgical sponges (see, e.g., U.S. Pat. Nos.3,956,044 and 4,045,238; available from Weck, Alcon, and Mentor), thathas been soaked in the composition and that releases the compositionupon contact with the host tissue. These are particularly useful forapplication to NS lesion sites during surgery in which only a singleadministration is possible. The compositions may also be applied inpellets (such as Elvax pellets, made of ethylene-vinyl acetate copolymerresin; about 0.5-100, preferably 1-20, and more preferably 1-5 μg ofconjugate per 1 mg resin) that can be implanted in the vicinity of thelesion during surgery.

In preferred embodiments, it is contemplated that a biodegradable matrixwill likely be most useful. A biodegradable matrix is generally definedas one that is capable of being reabsorbed into the body. Potentialbiodegradable matrices for use in connection with the compositions,devices and methods of this invention include, for example,biodegradable and chemically defined calcium sulfate,tricalciumphosphate, hydroxyapatite, polyactic acid, polyanhydrides,matrices of purified proteins, and semipurified extracellular matrixcompositions.

Other biocompatible biodegradable polymers that may be used are wellknown in the art and include, by way of example and not limitation,polyesters such as polyglycolides, polylactides and polylacticpolyglycolic acid copolymers (“PLGA”) (Langer and Folkman, 1976, Nature263:797-800); polyethers such as polycaprolactone (“PCL”);polyanhydrides; polyalkyl cyanoacrylates such as n-butyl cyanoacrylateand isopropyl cyanoacrylate; polyacrylamides; poly(orthoesters);polyphosphazenes; polypeptides; polyurethanes; and mixtures of suchpolymers.

It is to be understood that virtually any polymer that is now known orthat will be later developed and that may be suitable for the sustainedor controlled release of nucleic acids may be employed in the presentinvention.

In preferred embodiments, the biocompatible biodegradable polymer is acopolymer of glycolic acid and lactic acid (“PLGA”) having a proportionbetween the lactic acid/glycolic acid units ranging from about 100/0 toabout 25/75. The average molecular weight (“MW”) of the polymer willtypically range from about 6,000 to 700,000 and preferably from about30,000 to 120,000, as determined by gel-permeation chromatography usingcommercially available polystyrene of standard molecular weight, andhave an intrinsic viscosity ranging from 0.5 to 10.5.

The length of the period of continuous sustained or controlled releaseof nucleic acids from the matrix according to the invention will dependin large part on the MW of the polymer and the composition ratio oflactic acid/glycolic acid. Generally, a higher ratio of lacticacid/glycolic acid, such as for example 75/25, will provide for a longerperiod of controlled of sustained release of the nucleic acids, whereasa lower ratio of lactic acid/glycolic acid will provide for more rapidrelease of the nucleic acids. Preferably, the lactic acid/glycolic acidratio is 50/50.

The length of period of sustained or controlled release is alsodependent on the MW of the polymer. Generally, a higher MW polymer willprovide for a longer period of controlled or sustained release. In thecase of preparing, for example, matrices providing controlled orsustained release for about three months, when the composition ratio oflactic acid/glycolic acid is 100/0, the preferable average MW of polymerranges from about 7,000 to 25,000; when 90/10, from about 6,000 to30,000; and when 80/20, from about 12,000 to 30,000.

Another type of biomaterial that may be used is small intestinalsubmucosa (SIS). The SIS graft material may be prepared from a segmentof jejunum of adult pigs. Isolation of tissue samples may be carried outusing routine tissue culture techniques such as those described inBadybak et al., J. Surg. Res. 47:74-80, 1989. SIS material is preparedby removal of mesenteric tissue, inversion of the segment, followed byremoval of the mucosa and superficial submucosa by a mechanical abrasiontechnique. After returning the segment to its original orientation, theserosa and muscle layers are rinsed and stored for further use.

Another particular example of a suitable material is fibrous collagen,which may be lyophilized following extraction and partial purificationfrom tissue and then sterilized. Matrices may also be prepared fromtendon or dermal collagen, as may be obtained from a variety ofcommercial sources, such as, e.g., Sigma and Collagen Corporation.Collagen matrices may also be prepared as described in U.S. Pat. Nos.4,394,370 and 4,975,527, each incorporated herein by reference.

In addition, lattices made of collagen and glycosaminoglycan (GAG) suchas that described in Yannas & Burke, U.S. Pat. No. 4,505,266, may beused in the practice of the invention. The collagen/GAG matrix mayeffectively serve as a support or “scaffolding” structure into whichrepair cells may migrate. Collagen matrices, such as those disclosed inBell, U.S. Pat. No. 4,485,097, may also be used as a matrix material.

The various collagenous materials may also be in the form of mineralizedcollagen. For example, the fibrous collagen implant material termedUltraFiber™, as may be obtained from Norian Corp. (1025 Terra BellaAve., Mountain View, Calif., 94043), may be used for formation ofmatrices. U.S. Pat. No. 5,231,169, incorporated herein by reference,describes the preparation of mineralized collagen through the formationof calcium phosphate mineral under mild agitation in situ in thepresence of dispersed collagen fibrils. Such a formulation may beemployed in the context of delivering a nucleic acid segment to acentral nervous system site.

At least 20 different forms of collagen have been identified and each ofthese collagens may be used in the practice of the invention. Forexample, collagen may be purified from hyaline cartilage, as isolatedfrom diarthrodial joints or growth plates. Type II collagen purifiedfrom hyaline cartilage is commercially available and may be purchasedfrom, e.g., Sigma Chemical Company, St. Louis. Type I collagen frombovine tendon may be purchased from, e.g., Collagen Corporation. Asanother example, autologous extracellular matrix material, including butnot limited to products of biopsy explants cultivated ex vivo, may alsobe prepared from patient tissue for production of GAM. (See, e.g., U.S.Pat. No. 5,332,802 and references cited therein; West et al., Dermatol.Surg. 24:510-512, 1998; Staskowski et al., Otolaryngol Head Neck Surg.118(2):187-190, 1998; Rogalla, Minim Invasive Surg. Nurs. 11(2):67-69,1997.) Any form of recombinant collagen may also be employed, as may beobtained from a collagen-expressing recombinant host cell, includingbacterial yeast, mammalian, and insect cells. When using collagen an amatrix material it may be advantageous to remove what is referred to asthe “telopeptide” which is located at the end of the collagen moleculeand is known to induce an inflammatory response.

GAM may also be produced using fibrin matrices, the formation of whichcan be induced by contacting thrombin with a plasma protein fractioncontaining fibrinogen and factor XIII. The use of these plasmacomponents to produce biocompatible matrices is well known, and may beprovided, for example, by the TISEEL™ kit available from Immuno AG(Vienna, Austria). The person having ordinary skill in the art will befamiliar with these and other matrix materials suitable for making GAMswithin the scope and spirit of the present invention.

d. Preparation of the Gene Activated Matrices

In preferred embodiments, compositions of either or both biological andsynthetic matrices and DNA may be lyophilized together to form a drypharmaceutical powder. The gene activated matrix may be rehydrated priorto implantation in the body, or alternatively, the gene activated matrixmay become naturally rehydrated when placed in the body. The amount ofDNA, and the amount of contact time required for incorporation of theDNA into the matrix, will depend on the type of matrix used and can bereadily determined by one of ordinary skill in the art without undueexperimentation. Alternatively, the DNA may be encapsulated within amatrix of synthetic polymers, such as, for example, block copolymers ofpolylactic-polyglycolic acid (See Langer and Folkman, Nature263:797-800, 1976, which is incorporated herein by reference). Again,these parameters can be readily determined by one of ordinary skill inthe art without undue experimentation. For example, the amount of DNAconstruct that is applied to the matrix will be determined consideringvarious biological and medical factors. One would take intoconsideration the particular gene, the matrix, the site of the wound,the mammalian host's age, sex and diet and any further clinical factorsthat may effect wound healing such as the serum levels of variousfactors and hormones.

In additional embodiments of the invention, matrix or implant materialis contacted with the DNA encoding a therapeutic product of interest bysoaking the matrix material in a recombinant DNA stock solution.

In some instances medical devices such as implants, sutures, wounddressings, etc. may be coated with the nucleic acid compositions of theinvention using conventional coating techniques as are well known in theart. Such methods include, by way of example and not limitation, dippingthe device in the nucleic acid composition, brushing the device with thenucleic acid composition and/or spraying the device with the aerosolnucleic acid compositions of the invention. The device is then dried,either at room temperature or with the aid of a drying oven, optionallyat reduced pressure. A preferred method for coating sutures is providedin the examples.

For sutures coated with a polymeric matrix containing plasmid DNA,applicants have discovered that applying a coating compositioncontaining a total of about 0.01 to 10 mg plasmid DNA and preferablyabout 1 to 5 mg plasmid DNA, to a 70 cm length of suture using about 5to 100, preferably about 5 to 50, and more preferably about 15 to 30coating applications yields a therapeutically effective and uniformcoating.

In a particularly preferred embodiment, the invention provides coatedsutures, especially sutures coated with a polymeric matrix containingnucleic acids encoding therapeutic proteins that stimulate wound healingin vivo.

In another particularly preferred embodiment, a viable cell isintroduced or incorporated into the GAM as a support cell. Withoutwishing to be bound by theory, the presence of a support cell as acomponent of a GAM may in certain situations influence the ability ofthe GAM to promote neuronal regeneration and/or neuronal survival, suchas may be desirable at an NS lesion site. Support cells that may beuseful according to this embodiment of the invention include but neednot be limited to Schwann cells, oligodendrocytes, astrocytes,microglial cells, fibroblasts, macrophages or inflammatory cells such asmacrophages, neutorphils, monocytes, granulocytes and lymphocytes. Thosefamiliar with the art will appreciate that in various wound healingcontexts including those involving NS, these and other support cells mayplay a contributory role in the generation of a favorable environmentfor promoting neuronal survival and/or axonal generation and/or axonalregeneration. A GAM containing support cells may also be referred toherein as a mixed GAM.

Sutures which may be coated in accordance with the methods andcompositions of the present invention include any suture of natural orsynthetic origin. Typical suture materials include, by way of exampleand not limitation, silk; cotton; linen; polyolefins such aspolyethylene and polypropylene; polyesters such as polyethyleneterephthalate; homopolymers and copolymers of hydroxycarboxylic acidesters; collagen (plain or chromicized); catgut (plain or chromicized);and suture-substitutes such as cyanoacrylates. The sutures may take anyconvenient form such as braids or twists, and may have a wide range ofsizes as are commonly employed in the art.

The advantages of coated sutures, especially sutures coated with apolymeric matrix containing nucleic acids encoding therapeutic proteinsthat stimulate wound healing or inhibit fibrosis cover virtually everyfield of surgical use in humans and animals.

e. Uses of the Gene Activated Matrix

The GAM is applicable to a wide variety of tissue repair and woundhealing situations in human medicine. These include, but are not limitedto, regeneration of NS neural connections at lesion sites and may alsoinclude bone repair, tendon repair, ligament repair, blood vesselrepair, skeletal muscle repair, and skin repair. For example, using thegene activated matrix technology, neuronal therapeutic factors may besynthesized in axotomized neurons that have been transfected byretrograde axonal delivery of neuronal therapeutic agent encoding genesrecovered from a GAM. The therapeutic agents may direct ordered neuriteextension along axonal projection tracts, leading to reestablishment ofneural connections to distal targets. Such connections may in turnrestore the retrograde flow of neurotrophic factors to the perikaryonupon which neuronal networks depend. The end result is the augmentationof tissue repair and regeneration.

The GAM also may be useful when the clinical goal is to block a diseaseprocess, thereby allowing natural tissue healing to take place.Alternatively, the GAM may be used to replace a genetically defectiveprotein function, or to promote neuronal axon regeneration instead ofscar matrix deposition that might otherwise occur in the course ofnatural tissue remodeling without clinical intervention.

NS lesions may arise from traumatic/contusion-compression, transectionor other physical injury, or alternatively, from tissue damage eitherinduced by, or resulting from, a surgical procedure, from vascularpharmacologic or other insults including hemorrhagic or ischemic damage,or from neurodegenerative or other neurological diseases. The geneactivated matrix of the invention can be transferred to the patientusing various techniques. For example, matrices can be transferreddirectly to the site of the wound by the hand of the physician, eitheras a therapeutic implant or as a coated device (e.g., suture, coatedimplant, etc.).

The process of wound healing is a coordinated sequence of events whichincludes, hemorrhage, clot formation, dissolution of the clot withconcurrent removal of damaged tissue, and deposition of granulationtissue as initial repair material. The granulation tissue is a mixtureof fibroblasts and capillary blood vessels. The wound healing processinvolves diverse cell populations including endothelial cells, stemcells, macrophages and fibroblasts. The regulatory factors involved inwound repair are known to include systemic hormones, cytokines, enzymes,growth factors, extracellular matrix proteins and other proteins thatregulate growth and differentiation.

One important feature of the present invention is that the formation ofscar tissue at the site of the wound may be regulated by the selectiveuse of gene activated matrices. The formation of scar tissue may beregulated by controlling the levels of therapeutic protein expressed,for example, by using GAMs containing DNA constructs encoding negativeregulators of granulation tissue (scar) deposition. In cases oftraumatic NS damage it is especially desirable to inhibit the formationof scar tissue to permit axonal regrowth along projection tracts and todiscourage localized accumulations of neurotrophic factors.

The methods of the present invention include the grafting ortransplantation of the matrices containing the DNA of interest into thehost. Procedures for transplanting the matrices may include surgicalplacement, or injection, of the matrices into the host. In instanceswhere the matrices are to be injected, the matrices are drawn up into asyringe and injected into a patient at the site of the lesion. Multipleinjections may be made at such sites. Alternatively, the matrices may besurgically placed at the site of the lesion. The amount of matricesneeded to achieve the purpose of the present invention i.e., stimulationof NS axonal regeneration, is variable depending on the size, age andweight of the host.

According to the present invention, when a gene activated matrix istransferred to a host, for example, by injection, implantation orsurgery, axonal regenerative activity is preferably sufficient enough tofacilitate neuron-GAM interaction. This is a preferred condition forinduction of the delivery of agents for neuronal regeneration andsurvival by retrograde axonal transport. In the absence of such ongoingaxonal regenerative activity, it is within the scope of the invention toprovide agents that stimulate neurons to encourage neuron-GAMinteraction and promote axonal uptake of therapeutic constructs and/orcomplexes. Such stimulatory agents are known in the art and may includeagents that specifically stimulate neurons (e.g., neurotrophins) andagents that non-specifically promote any cellular uptake of complexes,including but not limited to inducers of membrane permeability; inducersof endocytic, plasma membrane biogenesis and recycling activities;ionophores, channel blockers and membrane depolarizing agents; signaltransduction molecules, gene activators, metalloproteases or any otheragent that may transiently rescue an injured neuron that is not activelyengaged in axonal regeneration. Physical or mechanical intervention mayalso effect neuron-GAM interaction, induction of axonal regenerativeactivity and/or axonal uptake of therapeutic constructs or complexes,including, for example, resection of the nerve tract proximal to theoriginal lesion site to restimulate regeneration. In any case, axonalregenerative activity that leads to GAM invasion by the growing axon maybe a preferred embodiment of the present invention.

Conduits

A conduit or nerve regeneration channel may be formulated using anybiocompatible matrix material containing DNA encoding a therapeuticagent of interest as described herein, for example therapeutic proteins,transcriptional products, antisense nucleic acids or ribozymes, and usedin accordance with the invention. The device may be formed so as toreceive one or more ends of a severed or damaged nerve, for example,from either side of a lesion point. The conduit, for example a tubularsemipermeable device, a hollow cylinder or a device having some otherconfiguration that those skilled in the art will appreciate as suitablefor a particular use of the conduit, defines a lumen through which axonsmay regenerate, including regeneration that leads to reestablishment ofneural networks and restoration of motor and/or sensory function, asdescribed herein. The conduit allows the diffusion or dispersion, interalia, of nutrients, metabolites and/or the gene-activated matrix itselfto the regenerating nerve site while excluding fibroblasts and othercells that may result in the formation of scar tissue. The conduit,comprising a gene activated matrix as described herein, guides neuronoutgrowth from a proximal damaged site to a distal damaged site, thusproviding effective enervation of the distal site.

In certain embodiments, the conduit may be multilayered and maycomprise, wholly or in part, gene activated matrix material. In oneembodiment, the conduit comprising gene activated matrices of theinvention may be derived from any biocompatible material. Such materialsmay include, but are not limited to, bioabsorbable or non-bioabsorbablematerials. The conduit may be derived from bioabsorbable polymers ornaturally occurring protein, for example, type I collagen, laminin,polyglycolic acid, glycolide trimethylene carbonate (GTMC), poly(L-lactide-co-6-caprolactone), glycoproteins, proteoglycans, heparansulfate proteoglycan, nidogen, glycosaminoglycans, fibronectin,epidermal growth factor, fibroblast growth factor, nerve growth factor,cytokines, DNA encoding growth factors or cytokines, or combinationsthereof.

In a further embodiment, the conduit comprising gene activated matricesof the invention may be derived from non-bioabsorbable syntheticpolymers, for example polyamide, polyimide, polyurethane, segmentedpolyurethane, polycarbonate, or silicone. Furthermore, the conduit ofthe present invention may be comprised of polyamide (nylon) filamentsinside silicone tubes. The conduit of the present invention may befurther comprised of a microporous synthetic polymer surface etched by alaser. In other further embodiments, the conduit comprising geneactivated matrices of the invention may be derived from interposed nervesegments and silicone tube conduit.

In certain other embodiments, the conduit comprising gene activatedmatrices of the invention may be derived from autogenous or autologousveins that are modified to serve as nerve conduits. According to certainof these embodiments, adventitial wall of the vein combined with geneactivated matrix promotes nerve regeneration by providing, inter alia,collagen, laminin, and/or Schwann cells, and promotes increasedvascularization of the new nerve. Alternatively, a conduit comprisinggene activated matrices of the invention may be derived from collagen,laminin, and Schwann cells.

The conduit may be formulated essentially as described for the geneactivated matrix of the present invention, including composition andpore size of the walls. The conduit may be of any shape, dimension, sizeor configuration, regular or irregular, according to the particular useand/or anatomical location intended. Preferably, the conduit willcomprise a lumen having an inner diameter of from about 1 mm to about 1cm. a wall diameter of from about 0.05 mm to about 1.0 mm, and a lengthranging from several millimeters to several centimeters, depending onthe extent of the nerve injury.

In a further embodiment, the conduit may be multilayered. A multilayeredconduit comprises (1) an inner layer comprising a gene activated matrixwith a pore size in the range of from about 0.006 μm to about 5.0 μmthat selectively allows the diffusion of DNA encoding neuronaltherapeutic factors, while preventing infiltration, invasion ordiffusion of fibroblasts and/or other scar-forming cells; and (2) asubstantially porous outer layer.

Further descriptions of conduits are contained in U.S. Pat. Nos.4,877,029, 4,962,146, 5,019,087, and 5,026,381, each of which is hereinincorporated by reference in its entirety.

2. Nucleic Acid-Containing Constructs and Compositions

a. Therapeutic DNA

The present invention provides compositions and methods for NS neuronalprotection, survival and regeneration via axonal delivery of therapeuticDNAs, as described above. DNA molecules that encode therapeuticproducts, which are also referred to herein as neuronal therapeuticencoding agents, may in certain embodiments be axonally delivered andretrogradely transported to the cell body of the neuron, as alsodescribed above. According to the present invention, neuronaltherapeutic encoding agents are delivered to the neuronal axon, or tonon-neuronal cell types that can contribute to NS repair, via a geneactivated matrix. The neuronal therapeutic encoding agent thus comprisesan inactive prodrug that is transcribed and translated within a neuronalcell, to produce an active neuronal therapeutic agent, for example aneurotrophic protein factor. The active neuronal therapeutic agent(e.g., neurotrophic factor) stimulates axonal outgrowth into the geneactivated matrix, which may then deliver more neuronal therapeuticencoding agent (e.g., therapeutic DNA prodrug) that is expressed toprovide additional active agent. Upon activation of the growth response,neurons may secrete matrix degrading enzymes to facilitate axonalregrowth through the wound. By using a GAM to deliver to the lesion sitea neuronal therapeutic encoding agent instead of a neuronal therapeuticagent (such as a neurotrophic factor), the present invention thusovercomes problems in the prior art relating to axonal entrapment, byreducing or eliminating the formation of neurotrophic factor sinks.

Molecules that encode therapeutic products, which are also referred toherein as neuronal therapeutic agent encoding nucleic acids, aremolecules that effect a treatment upon or within a neuronal cell,generally by modifying gene transcription of translation. Therapeuticnucleic acids of the present invention may be used in the context of“positive” or “negative” gene therapy, depending on the effect one seeksto achieve.

For example, a therapeutic nucleotide sequence may encode all or aportion of a gene. If it encodes all (or the most critical functionalportions) of a gene, it may effect genetic therapy by serving as areplacement for a defective gene. Such a sequence may also function byrecombining with DNA already present in a cell, thereby replacing adefective portion of a gene.

A variety of positive gene therapy applications and therapeutic geneproducts are described herein and include such diverse applications asthe promotion of wound healing, the stimulation of neuronal survival andaxonal generation/regeneration, and the like. The replacement of adefective or nonfunctional gene with one that produces the desired geneproduct is also considered “positive” gene therapy, whether one isreplacing a dysfunctional or nonfunctional regulatory sequence or asequence that encodes a structural protein.

Similarly, “negative” gene therapy is encompassed by the presentinvention as well. Thus, therapeutic nucleic acids of the presentinvention may encode products that, for example, inhibit fibrosis,extracellular matrix deposition and/or scar tissue formation.Therapeutic nucleic acids, including neuronal therapeutic encodingagents of the present invention, may also encode decorin, a proteoglycanknown to inhibit TGF-β1. In a rat model of glomerulonephritis, fibrosisis mediated by TGF-11. In a gene therapy application, delivering decorincDNA to the muscle results in a marked therapeutic effect on fibrosisinduced by glomerulonephritis (Isaka et al., 1996, Nature Medicine2:418-423).

Further details regarding both positive and negative gene therapyapplications are set forth below in subsequent sections of thespecification. The following illustrations are thus intended to beexemplary and not limiting.

i. DNA Encoding Neurotrophic Agents

(a) Neuronal Therapeutic Encoding Agents

Nucleic acids for delivery include nucleic acid molecules that encodeneuronal therapeutic agents, which may further include proteins topromote neuronal growth and/or survival. For example, in NS injuryneuronal cells may fail to regenerate axons over a sufficient distanceto re-establish neural connections and restore the retrograde deliveryof neurotrophic factors from distal neuronal targets to the perikarya. Aconstruct that directs neuronal expression of one or more neurotrophins,alone or in combination with neurotrophin or FGF protein to promoteshort-term neuronal survival, can be used to combat the effects ofaxotomy.

Examples of neuronal therapeutic encoding agents include growth factorsand neurotrophic agents that promote neuronal growth and/or survival.Such examples include, but are not limited to, nerve growth factor(NGF), brain-derived neurotrophic factor (BDNF), cardiotrophin-1 (CT-1),choline acetyltransferase development factor (CDF), ciliary neurotrophicfactor (CNTF) fibroblast growth factor-1 (FGF-1), FGF-2, FGF-5, glialcell-line-derived neurotrophic factor (GDNF), insulin, insulin-likegrowth factor-1 (IGF-1), IGF-2, interleukin-6 (IL-6), leukemia inhibitorfactor (LIF), neurite promoting factor (NPF), neurotrophin-3 (NT-3),NT-4, platelet-derived growth factor (PDGF), protease nexin-1 (PN-1),S-100, transforming growth factory (TGF-β), decorin, anti-TGF-betaantibodies, mutated TGF-beta, and vasoactive intestinal peptide (VIP).(Oppenheim, 1996, Neuron 17:195-197.)

The neuronal therapeutic-encoding genes of the present invention mayinclude genes that encode neuronal therapeutic agents that are secreted,or that are not secreted, or that are targeted for localization tospecific subcellular compartments within the cell. Nucleic acidsequences encoding peptides that direct intracellular sorting of newlysynthesized polypeptides to secretory pathways or to residence inparticular intracellular compartments are known and are within the scopeof the present invention.

Thus, for example, nucleic acid constructs that are neuronaltherapeutic-encoding agents may contain sequences encoding peptides thatdirect an encoded neuronal therapeutic agent to be retained in thecytosol, to reside in the lumen of the endoplasmic reticulum (ER), to besecreted from a cell via the classical ER-Golgi secretory pathway, to beincorporated into the plasma membrane, to associate with a specificcytoplasmic component including the cytoplasmic domain of atransmembrane cell surface receptor or to be directed to a particularsubcellular location by a known intracellular protein sorting mechanismwith which those skilled in the art will be familiar. Such intracellularprotein sorting peptide sequences may also be present in ligands ornucleic acid binding domains that are provided by the present invention.

In one embodiment of this aspect of the invention, neuronal therapeuticencoding agents may be nucleic acid molecules that encode neuronaltherapeutic agents that are ordinarily secretory proteins, but fromwhich sequences encoding secretory “signal” peptides have been deletedto prevent such neuronal therapeutic agents from being secreted via theclassical ER-Golgi protein secretory pathway.

Without wishing to be bound by theory, such neuronal therapeuticencoding agents are believed to encode neuronal therapeutic agents thatmay be useful in the present invention because they may not be secretedby cells expressing the delivered nucleic acids. Such agents may beparticularly useful to overcome the problem of extracellularneurotrophic factor sinks that give rise to entrapment of regeneratingaxons, as described above. Such agents may also be useful where they mayexert their neurotrophic effects via intracellular interactions withneuronal components. In this scheme, the agents provided by the presentinvention may reflect a departure from currently accepted models ofneurotrophic factor activity, which require binding interaction betweenan extracellular neurotrophic factor and an exteriorly disposed neuronalcell surface receptor.

Neuronal therapeutic agents lacking secretory signal sequences and thatare the expressed products of neuronal therapeutic-encoding agentsdelivered to cells according to the present invention may further beuseful where the ligand may be bound and internalized by both neuronaland non-neuronal cell types, neither of which is capable of secretingthe expressed neuronal therapeutic agent, but where the neuronaltherapeutic agent as an intracellular component may exert onlyneurotrophic/neuronal therapeutic effects that promote axonalregeneration. According to this non-limiting model, cell surfacereceptors for ligands of the invention need not be absolutely restrictedin their expression to neuronal cell surfaces, because non-neuronalcells at or near a NS lesion site would not be able to secrete theencoded neuronal therapeutic agents and therefore cannot generateneurotrophic factor sinks that can lead to undesirable axonalentrapment, as described above. These and other advantages of neuronaltherapeutic-encoding agents lacking nucleic acid sequences that encodesecretory signal sequences will be appreciated by those skilled in theart.

(b) Other Neuronal Therapeutic Agents

In the context of treatment of neurons following NS injury that mayresult from physical injury, neurological diseases or neurodegenerativediseases including autoimmune and/or inflammatory diseases, or othertrauma, it may be useful to specifically inhibit or interfere withcertain biological responses to such injury. For example, as describedabove, various cell types in an affected tissue may participate infibrotic scar deposition that may, inter alia, lead to undesirablegrowth factor sinks and may further present impediments to NSregeneration and reestablishment of neural networks.

As another example, in neurodegenerative disease central nervous system(CNS) injury wherein CNS microglia contribute to the pathogenesis,neuronal therapeutic agents that are targeted to and capable ofregulating the biological activity of such microglia may be useful. Forinstance, neuronal therapeutic agents that are targeted to regulate theviability, biosynthetic potential or proliferative capacity of, e.g.,microglia, or neuronal therapeutic encoding agents that deliver genesable to regulate one or more pathogenic gene products of, e.g.,microglia, are non-limiting illustrations of additional agents accordingto the invention that may be useful. Examples of target microglia geneproducts that may impede reestablishment of neural connectivityfollowing CNS and or NS injury include but need not be limited to TGF-β,connective tissue growth factor (CTGF), IL-1 receptor antagonist(IL-IRA) (see, e.g., Streit, In CNS Injuries: Cellular Responses andPharmacological Strategies, M. Berry and A. Logan, eds., 1998, CRCPress, Boca Raton, Fla.), macrophage/microglial stimulatory factor(MSF), macrophage/microglial inhibitory factor (MIF) andmicroglia-derived proteases (e.g., metalloproteases, plasminogenactivator).

It should expressly be understood, however, that simply because a cellor tissue is described herein as “targeted” does not necessarily implythat a targeting ligand is a required component of a therapeuticconstruct according to the present invention. Therapeutic nucleotidesequences/constructs are deliverable in a variety of forms, as disclosedherein, e.g., in the presence of—or absence of—a targeting ligand.

The constructs provided herein may also be used to deliver a ribozyme,antisense molecule, and the like to targeted cells, for example, tospecifically inhibit activation of one or more genes following NSinjury. These nucleic acids may be present in the complex of ligand andnucleic acid binding domain or encoded by a nucleic acid in the complex.Alternatively, the nucleic acid may be directly linked to the ligand.Such products include antisense RNA, antisense DNA, ribozymes,triplex-forming oligonucleotides, and oligonucleotides that bindproteins. The nucleic acids can also include RNA trafficking signals,such as viral packaging sequences (see, e.g., Sullenger et al. (1994)Science 262:1566-1569).

Nucleic acids and oligonucleotides for use as described herein can besynthesized by any method known to those of skill in this art (see,e.g., WO 93/01286, U.S. application Ser. No. 07/723,454; U.S. Pat. No.5,218,088; U.S. Pat. No. 5,175,269; U.S. Pat. No. 5,109,124).Identification of oligonucleotides and ribozymes for use as antisenseagents and DNA encoding genes for delivery for genetic therapy involvemethods well known in the art. For example, the desirable properties,lengths and other characteristics of such oligonucleotides are wellknown. Antisense oligonucleotides are typically designed to resistdegradation by endogenous nucleolytic enzymes by using such linkages as:phosphorothioate, methylphosphonate, sulfone, sulfate, ketyl,phosphorodithioate, phosphoramidate, phosphate esters, and other suchlinkages (see, e.g., Agrwal et al., Tetrehedron Lett. 28:3539-3542(1987); Miller et al., J. Am. Chem. Soc. 93:6657-6665 (1971); Stec etal., Tetrehedron Lett. 26:2191-2194 (1985); Moody et al., Nucl. AcidsRes. 12:4769-4782 (1989); Uznanski et al., Nucl. Acids Res. (1989);Letsinger et al., Tetrahedron 40:137-143 (1984); Eckstein, Annu. Rev.Biochem. 54:367-402 (1985); Eckstein, Trends Biol. Sci. 14:97-100(1989); Stein In: Oligodeoxynucleotides. Antisense Inhibitors of GeneExpression, Cohen, Ed, Macmillan Press, London, pp. 97-117 (1989); Jageret al., Biochemistry 27:7237-7246 (1988)).

Antisense nucleotides are oligonucleotides that bind in asequence-specific manner to nucleic acids, such as mRNA or DNA. Whenbound to mRNA that has complementary sequences, antisense preventstranslation of the mRNA (see, e.g., U.S. Pat. No. 5,168,053 to Altman etal.; U.S. Pat. No. 5,190,931 to Inouye, U.S. Pat. No. 5,135,917 toBurch; U.S. Pat. No. 5,087,617 to Smith and Clusel et al. (1993) Nucl.Acids Res. 21:3405-3411, which describes dumbbell antisenseoligonucleotides). Triplex molecules refer to single DNA strands thatbind duplex DNA forming a colinear triplex molecule, thereby preventingtranscription (see, e.g., U.S. Pat. No. 5,176,996 to Hogan et al., whichdescribes methods for making synthetic oligonucleotides that bind totarget sites on duplex DNA).

Particularly useful antisense nucleotides and triplex molecules aremolecules that are complementary to or bind the sense strand of DNA ormRNA that encodes a protein involved in neuronal cell degeneration(e.g., proteins of apoptosis pathways) or a protein mediating any otherunwanted process such that inhibition of translation of the protein isdesirable.

A ribozyme is a molecule that specifically cleaves RNA substrates, suchas mRNA, resulting in inhibition or interference with cell growth orexpression. There are at least five known classes of ribozymes involvedin the cleavage and/or ligation of RNA chains. Ribozymes can be targetedto any RNA transcript and can catalytically cleave such transcript (see,e.g., U.S. Pat. No. 5,272,262; U.S. Pat. No. 5,144,019; and U.S. Pat.Nos. 5,168,053, 5,180,818, 5,116,742 and 5,093,246 to Cech et al.). Anysuch ribozyme or nucleic acid encoding the ribozyme may be delivered toa cell via the use of a construct as disclosed herein.

Ribozymes, deoxyribozymes and the like may be delivered to the treatedor targeted cells by DNA encoding the ribozyme linked to a eukaryoticpromoter, such as a eukaryotic viral promoter, such that uponintroduction into the nucleus, the ribozyme will be directlytranscribed. Ribozyme-containing constructs may further comprise atargeting ligand and/or a nuclear translocation sequence. The latter maybe included as part of the ligand, as part of a linker between theligand and nucleic acid binding domain, or it may be attached directlyto the NABD.

ii. Methods of Preparing DNA for Use in Compositions

A therapeutic nucleotide composition, which may be a neuronaltherapeutic encoding agent of the present invention, comprises anucleotide sequence encoding a therapeutic molecule as described herein.As noted above, a therapeutic nucleotide composition or neuronaltherapeutic encoding agent may further comprise an enhancer element or apromoter located 5′ to and controlling the expression of saidtherapeutic nucleotide sequence or gene. The promoter is a DNA segmentthat contains a DNA sequence that controls the expression of a genelocated 3′ or downstream of the promoter. The promoter is the DNAsequence to which RNA polymerase specifically binds and initiates RNAsynthesis (transcription) of that gene, typically located 3′ of thepromoter.

The subject therapeutic nucleotide composition comprises a nucleic acidmolecule, which in certain aspects of the invention further comprises atleast 2 different operatively linked DNA segments. The DNA can bemanipulated and amplified by PCR and by using the standard techniquesdescribed in Molecular Cloning: A Laboratory Manual, 2nd Edition,Maniatis et al., eds., Cold Spring Harbor, N.Y. (1989). Typically, toproduce a therapeutic nucleotide composition of the present invention,the sequence encoding the selected therapeutic composition and thepromoter or enhancer are operatively linked to a vector DNA moleculecapable of autonomous replication in a cell either in vivo or in vitro.By operatively linking the enhancer element or promoter and thenucleotide sequence encoding the therapeutic nucleotide composition tothe vector, the attached segments are replicated along with the vectorsequences. Thus, a recombinant DNA molecule (rDNA) of the presentinvention is a hybrid DNA molecule comprising at least 2 nucleotidesequences not normally found together in nature.

The therapeutic nucleotide composition of the present invention is fromabout 20 base pairs to about 100,000 base pairs in length. Preferablythe nucleic acid molecule is from about 50 base pairs to about 50,000base pairs in length. More preferably the nucleic acid molecule is fromabout 50 base pairs to about 10,000 base pairs in length. Most preferredis a nucleic acid molecule from about 50 pairs to about 4,000 base pairsin length. The therapeutic nucleotide can be a gene or gene fragmentthat encodes a protein or peptide that provides the desired therapeuticeffect. Alternatively, the therapeutic nucleotide can be a DNA or RNAoligonucleotide sequence that exhibits enzymatic therapeutic activity.Examples of the latter include antisense oligonucleotides that willinhibit the transcription of deleterious genes or ribozymes that act assite-specific ribonucleases for cleaving selected mutated genesequences. In another variation, a therapeutic nucleotide sequence ofthe present invention may comprise a DNA construct capable of generatingtherapeutic nucleotide molecules, including ribozymes and antisense DNA,in high copy numbers in target cells, as described in published PCTapplication No. WO 92/06693 (the disclosure of which is incorporatedherein by reference). Exemplary and preferred nucleotide sequencesencode an expressible peptide, polypeptide or protein, and may furtherinclude an active constitutive or inducible promoter sequence.

A regulatable promoter is a promoter where the rate of RNA polymerasebinding and initiation is modulated by external stimuli. Such stimuliinclude compositions light, heat, stress and the like. Inducible,suppressible and repressible promoters are regulatable promoters.Regulatable promoters may also include tissue specific promoters. Tissuespecific promoters direct the expression of that gene to a specific celltype. Tissue specific promoters cause the gene located 3′ of it to beexpressed predominantly, if not exclusively in the specific cells wherethe promoter expressed its endogenous gene. Typically, it appears thatif a tissue-specific promoter expresses the gene located 3′ of it atall, then it is expressed appropriately in the correct cell types as hasbeen reviewed by Palmiter et al., Ann. Rev. Genet. 20:465-499 (1986).

When a tissue specific promoter controls the expression of a gene, thatgene will be expressed in a small number of tissues or cell types ratherthan in substantially all tissues and cell types. Examples of tissuespecific promoters include the glial fibrillary acid protein (GFAP) genepromoter (Brenner and Messing, 1996, Methods: A companion to Methods inEnzymology 10:351-364); the GAP43 promoter (deGroen et al., 1995, J.Mol. Neurosci, 66:109-119); the immunoglobulin promoter described byBrinster et al., Nature 306:332-336 (1983) and Storb et al., Nature310:238-231 (1984); the elastase-I promoter described by Swift et al.,Cell 38:639-646 (1984); the globin promoter described by Townes et al.,Mol. Cell. Biol. 5:1977-1983 (1985), and Magram et al., Mol. Cell. Biol.9:4581-4584 (1989), the insulin promoter described by Bucchini et al.,PNAS USA 83:2511-2515 (1986) and Edwards et al., Cell 58:161 (1989); theimmunoglobulin promoter described by Ruscon et al., Nature 314:330-334(1985) and Grosscheld et al., Cell 38:647-658 (1984); the alpha actinpromoter described by Shani, Mol. Cell. Biol. 6:2624-2631 (1986); thealpha crystalline promoter described by Overbeek et al., PNAS USA82:7815-7819 (1985); the prolactin promoter described by Crenshaw etal., Genes and Development 3:959-972 (1989); the proopiomelanocortinpromoter described by Tremblay et al., PNAS USA 85:8890-8894 (1988); thebeta-thyroid stimulating hormone (BTSH) promoter described by Tatsumi etal., Nippon Rinsho 47:2213-2220 (1989); the mouse mammary tumor virus(MMTV) promoter described by Muller et al., Cell 54:105 (1988); thealbumin promoter described by Palmiter et al., Ann. Rev. Genet.20:465-499 (1986); the keratin promoter described by Vassar et al., PNASUSA 86:8565-8569 (1989); the osteonectin promoter described by McVey etal., J. Biol. Chem. 263:11,111-11,116 (1988); the prostate-specificpromoter described by Allison et al., Mol. Cell. Biol. 9:2254-2257(1989); the opsin promoter described by Nathans et al., PNAS USA81:4851-4855 (1984); the olfactory marker protein promoter described byDanciger et al., PNAS USA 86:8565-8569 (1989); the neuron-specificenolase (NSE) promoter described by Forss-Pelter et al., J. Neurosci.Res. 16:141-151 (1986); the L-7 promoter described by Sutcliffe, Trendsin Genetics 3:73-76 (1987) and the protamine 1 promoter describedPeschon et al., Ann. New York Acad. Sci. 564:186-197 (1989) and Braun etal., Genes and Development 3:793-802 (1989).

In various alternative embodiments of the present invention, therapeuticsequences and compositions useful for practicing the therapeutic methodsdescribed herein are contemplated. Therapeutic compositions of thepresent invention may contain a physiologically tolerable carriertogether with one or more therapeutic nucleotide sequences of thisinvention, dissolved or dispersed therein as an active ingredient. In apreferred embodiment, the composition is not immunogenic or otherwiseable to cause undesirable side effects when administered to a mammal orhuman patient for therapeutic purposes.

As used herein, the terms “pharmaceutically acceptable”,“physiologically tolerable” and grammatical variations thereof, as theyrefer to compositions, carriers, diluents and reagents, are usedinterchangeably and represent that the materials are capable ofadministration to or upon a mammal without the production of undesirablephysiological effects such as nausea, dizziness, gastric upset and thelike.

The preparation of a pharmacological composition that contains activeingredients dissolved or dispersed therein is well understood in theart. Typically such compositions are prepared as injectables either asliquid solutions or suspensions, or as pastes, however, solid formssuitable for solution, or suspensions, in liquid prior to use can alsobe prepared. The preparation can also be emulsified, or formulated intopastes, suppositories, ointments, creams, dermal patches, or the like,depending on the desired route of administration.

The active ingredient can be mixed with excipients which arepharmaceutically acceptable and compatible with the active ingredientand in amounts suitable for use in the therapeutic methods describedherein. Suitable excipients are, for example, water, saline, dextrose,glycerol, ethanol or the like and combinations thereof, includingvegetable oils, propylene glycol, polyethylene glycol and benzyl alcohol(for injection or liquid preparations); and Vaseline, vegetable oil,animal fat and polyethylene glycol (for externally applicablepreparations). In addition, if desired, the composition can containwetting or emulsifying agents, isotonic agents, dissolution promotingagents, stabilizers, colorants, antiseptic agents, soothing agents andthe like additives (as usual auxiliary additives to pharmaceuticalpreparations), pH buffering agents and the like which enhance theeffectiveness of the active ingredient.

The therapeutic compositions of the present invention can includepharmaceutically acceptable salts of the components therein.Pharmaceutically acceptable salts include the acid addition salts(formed with the free amino groups of the polypeptide) that are formedwith inorganic acids such as, for example, hydrochloric or phosphoricacids, or such organic acids as acetic, tartaric, mandelic and the like.Salts formed with the free carboxyl groups can also be derived frominorganic bases such as, for example, sodium, potassium, ammonium,calcium or ferric hydroxides, and such organic bases as isopropylamine,trimethylamine, 2-ethylamino ethanol, histidine, procaine and the like.

Physiologically tolerable carriers are well known in the art. Exemplaryof liquid carriers are sterile aqueous solutions that contain nomaterials in addition to the active ingredients and water, or contain abuffer such as sodium phosphate at physiological pH value, physiologicalsaline or both, such as phosphate-buffered saline. Still further,aqueous carriers can contain more than one buffer salt, as well as saltssuch as sodium and potassium chlorides, dextrose, polyethylene glycoland other solutes. Physiologically tolerable carriers may also includecompositions that mimic relevant tissue fluids, e.g., artificialcerebral spinal fluid, or artificial blood.

Liquid compositions can also contain liquid phases in addition to and tothe exclusion of water. Exemplary of such additional liquid phases areglycerin, vegetable oils such as cottonseed oil, and water-oilemulsions.

A therapeutic composition typically contains an amount of a therapeuticnucleotide sequence of the present invention sufficient to deliver atherapeutically effective amount to the target tissue, typically anamount of at least 0.1 weight percent to about 90 weight percent oftherapeutic nucleotide sequence per weight of total therapeuticcomposition. A weight percent is a ratio by weight of therapeuticnucleotide sequence to total composition. Thus, for example, 0.1 weightpercent is 0.1 grams of DNA segment per 100 grams of total composition.

The therapeutic nucleotide compositions comprising syntheticoligonucleotide sequences of the present invention can be prepared usingany suitable method, such as, the phosphotriester or phosphodiestermethods. See Narang et al., Meth. Enzymol. 68: 90, (1979); U.S. Pat. No.4,356,270; and Brown et al., Meth. Enzymol. 68:109 (1979). Fortherapeutic oligonucleotides sequence compositions in which a family ofvariants is preferred, the synthesis of the family members can beconducted simultaneously in a single reaction vessel, or can besynthesized independently and later admixed in preselected molar ratios.

For simultaneous synthesis, the nucleotide residues that are conservedat preselected positions of the sequence of the family member can beintroduced in a chemical synthesis protocol simultaneously to thevariants by the addition of a single preselected nucleotide precursor tothe solid phase oligonucleotide reaction admixture when that positionnumber of the oligonucleotide is being chemically added to the growingoligonucleotide polymer. The addition of nucleotide residues to thosepositions in the sequence that vary can be introduced simultaneously bythe addition of amounts, preferably equimolar amounts, of multiplepreselected nucleotide precursors to the solid phase oligonucleotidereaction admixture during chemical synthesis. For example, where allfour possible natural nucleotides (A, T, G and C) are to be added at apreselected position, their precursors are added to the oligonucleotidesynthesis reaction at that step to simultaneously form four variants.

This manner of simultaneous synthesis of a family of relatedoligonucleotides has been previously described for the preparation of“degenerate oligonucleotides” by Ausubel et al, in Current Protocols inMolecular Biology, Suppl. 8:2.11.7, John Wiley & Sons, Inc., New York(1991), and can readily be applied to the preparation of the therapeuticoligonucleotide compositions described herein.

Nucleotide bases other than the common four nucleotides (A, T, G or C),or the RNA equivalent nucleotide uracil (U), can be used in the presentinvention. For example, it is well known that inosine (I) is capable ofhybridizing with A, T and G, but not C. Thus, where all four commonnucleotides are to occupy a single position of a family ofoligonucleotides, that is, where the preselected therapeutic nucleotidecomposition is designed to contain oligonucleotides that can hybridizeto four sequences that vary at one position, several differentoligonucleotide structures are contemplated. The composition can containfour members, where a preselected position contains A, T, G or C.Alternatively, the composition can contain two members, where apreselected position contains I or C, and has the capacity the hybridizeat that position to all four possible common nucleotides. Finally, othernucleotides may be included at the preselected position that have thecapacity to hybridize in a non-destabilizing manner with more than oneof the common nucleotides in a manner similar to inosine.

3. Testing of Constructs

The reprogrammed recombinant nucleic acid, synthetic DNA or viraldelivery vehicles may be assessed in any number of in vitro modelsystems. In particular, target cells are grown in culture and incubatedwith the nucleic acid delivery vehicle. The nucleic acid can encode areporter, in which case the reporter product is assayed, or a neuronaltherapeutic agent, in which case neuronal outgrowth, neurite extension,or another parameter for routinely determining neuronal therapeuticencoding agent expression with which those skilled in the art will befamiliar, is measured. Moreover, any assayable gene product can be used.For reporter genes, a wide variety of suitable genes are available. Suchreporters include β-galactosidase, alkaline phosphatase,β-glucuronidase, green fluorescent protein, luciferase, large Tantigenor any protein for which an antibody exists or can be developed.The choice of a reporter depends, in part, upon the cells being tested.Alternatively, the nucleic acid can encode a neuronal therapeutic agent.Such products include all those described herein.

The delivery vehicles may be assessed in in vitro or in vivo modelsystems. Generally, in vitro testing in relevant cultured neuronal cellsmay be used, e.g., retinal ganglion cells, dorsal root ganglion cells,neural progenitor cells or astrocytes. Furthermore, in vivo modelsystems may include, for example, optic nerve and spinal cord bioassaysas described herein or any suitable in vivo model neuronal system withwhich those having skill in the art are familiar.

a. Targeting Agents

Although the various DNA devices and constructs disclosed herein do notabsolutely require the inclusion of a targeting moiety, in variousembodiments, inclusion of a target ing agent—e.g., a polypeptideligand—may be advantageous. Examples of useful ligands are describedbelow for the purpose of illustrating such embodiments, but suchexamples should not be perceived as limiting the invention to suchembodiments alone.

i. Ligands

Ligands according to the present invention are molecules capable ofbinding interactions with receptors of desired target cells, and maytake a variety of forms. Ligands that are most preferred for use in theinvention are internalized by target cells subsequent to receptorbinding, providing a cellular route of entry for targeted agents of theinvention. Ligands may be natural or synthetic molecules and may besubunits, fragments or structurally modified forms of other ligands.Thus, ligands may include, but need not be limited to, proteins,peptides, polypeptides, muteins, fragments or chemical derivatives ofproteins, peptides, or polypeptides, other natural or syntheticmolecules such as carbohydrates, nucleic acids or their derivatives,lipids or their derivatives, or any other natural or artificialcomposition that binds to cellular receptors. In many aspects of theinvention, preferred ligands bind to receptors on the surfaces ofneuronal cells, but the invention need not be so limited.

(a) Ligands that Bind to and are Internalized by Neuronal Cells

As noted above, receptor-binding internalized ligands may be used todeliver nucleic acids, including a neuronal therapeutic-encoding agent,to a cell expressing an appropriate receptor on its cell surface.Numerous molecules that bind specific receptors have been identified andare suitable for use in the present invention. Such molecules includeneurotrophic and other neuronal therapeutic factors, which may furtherinclude—but which are not limited to—growth factors, cytokines, andantibodies.

Many growth factors and families of growth factors share structural andfunctional features and may be used in the present invention. Familiesof growth factors include neurotrophins (NT) such as NT-1, NT-2, NT-3and NT-4/5, where NT-1 is nerve growth factor (NGF) and NT-2 is brainderived neurotrophic factor (BDNF). Additional growth factor familiesinclude ciliary neurotrophic factor (CNTF) and related neuropoieticcytokines including leukemia inhibitory factor (LIF) and oncostatin M(OSM); fibroblast growth factors including FGF-1 through FGF-15;pleiotrophins including midkine (Li, Science 250:1690, 1990) and heparinbinding neurotrophic factor (HBNF, He et al., J. Neurosci. 18:3699-3707,1998); cell surface proteoglycans (see, e.g., Quarto et al., J. CellSci. 107:3201-3212, 1994); and the epidermal growth factor (EGF) family.These and other soluble factors, such as TGF-α (transforming growthfactor), TGF-β and related factors including glial cell line derivedneurotrophic factor (GDNF), insulin and insulin-like growth factors(IGF), HB-EGF, cholera toxin B subunit (CTB), neurotensin, bombesin,substance P, neurokinin, tachykinin and other neuropeptides also bind tospecific identified receptors on cell surfaces of the NS, includingneuronal cell surfaces and may be used in the present invention.

Antibodies that are specific to cell surface molecules expressed byneuronal cells are readily generated as monoclonal antibodies or aspolyclonal antisera, or may be produced as genetically engineeredimmunoglobulins that are designed using methods well known in the art tohave desirable properties. For example, by way of illustration and notlimitation, recombinant IgGs, chimeric fusion proteins havingimmunoglobulin derived sequences or “humanized” antibodies may all beused as ligands that bind to and are internalized by neuronal cellsaccording to the invention. Many such antibodies are readily availablefrom a variety of commercial and other sources (e.g., from American TypeCulture Collection, Rockville, Md.). Cytokines, including but notlimited to interleukins, chemokines, and interferons, may also havespecific receptors on one or more cell type found in the NS and may beused as described herein. These and other ligands are discussed in moredetail below.

Fragments of ligands described herein may be used within the presentinvention, so long as the fragment retains the ability to bind to theappropriate cell surface molecule. Likewise, ligands with substitutionsor other alterations, but which retain binding ability, may also beused. In general, a particular ligand refers to a polypeptide(s) havingan amino acid sequence of the native ligand, as well as modifiedsequences (e.g., having amino acid substitutions, deletions, insertionsor additions compared to the native protein) as long as the ligandretains the ability to bind to its receptor on a neuronal cell and beinternalized.

Ligands also encompass muteins that possess the ability to bind toreceptor expressing cells and be internalized. The muteins may not bepharmacologically active. Such muteins include, but are not limited to,those produced by replacing one or more of the “native” amino acidresidues in a ligand amino acid sequence with a different amino acid, asdescribed herein. Typically, such muteins will have conservative aminoacid changes. For example, if the ligand is a polypeptide sequenceencoding FGF2, a useful mutein may include a cysteine residue in placeof a serine residue. DNA encoding such muteins will, unless modified byreplacement of degenerate codons, hybridize under conditions of at leastlow stringency to native DNA sequence encoding the wild-type ligand.

DNA encoding a ligand may be prepared synthetically based on known aminoacid or DNA sequence, isolated using methods known to those of skill inthe art (e.g., PCR amplification), or obtained from commercial or othersources. DNA encoding a ligand may differ from “known” or “native”sequences by substitution of degenerate codons or by encoding differentamino acids. Differences in amino acid sequences, such as thoseoccurring among the homologous ligand of different species as well asamong individual organisms or species, are tolerated as long as theligand binds to its receptor. Ligands may be isolated from naturalsources or made synthetically, such as by recombinant means or chemicalsynthesis.

(1) Polypeptides Reactive with the FGF Receptor

One family of growth factors that may be used as ligands within thecontext of the present invention is the fibroblast growth factor (FGF)family. The members of the FGF family have a high degree of amino acidsequence similarities and common physical and biological properties,including the ability to bind to one or more FGF receptors.

This family of proteins includes FGFs designated FGF-1 (acidic FGF(aFGF)), FGF-2 (basic FGF (bFGF)), FGF-3 (int-2) (see, e.g., Moore etal., EMBO J. 5:919-924, 1986), FGF-4 (hst-1/K-FGF) (see, e.g., Sakamotoet al., Proc. Natl. Acad. Sci. USA 86:1836-1840, 1986; U.S. Pat. No.5,126,323), FGF-5 (see, e.g., U.S. Pat. No. 5,155,217), FGF-6 (hst-2)(see, e.g., published European Application EP 0 488 196 A2; Uda et al.,Oncogene 7:303-309, 1992), FGF-7 (keratinocyte growth factor) (KGF)(see, e.g., Finch et al., Science 245:752-755, 1985; Rubin et al., Proc.Natl. Acad. Sci. USA 86:802-806, 1989; and International Application WO90/08771), FGF-8 (see, e.g., Tanaka et al., Proc Natl. Acad. Sci. USA89:8528-8532, 1992); FGF-9 (see, Miyamoto et al., Mol. Cell. Biol.13:4251-4259, 1993); FGF-11 (WO 96/39507); FGF-13 (WO 96/39508); FGF-14(WO 96/39506); and FGF-15 (WO 96/39509).

DNA encoding FGF peptides and/or the amino acid sequences of FGFs arewell known. For example, DNA encoding human FGF-1 (Jaye et al., Science233:541-545, 1986; U.S. Pat. No. 5,223,483), bovine FGF-2 (Abraham etal., Science 233:545-548, 1986; Esch et al., Proc. Natl. Acad. Sci. USA82:6507-6511, 1985; and U.S. Pat. No. 4,956,455), human FGF-2 (Abrahamet al., EMBO J. 5:2523-2528, 1986; U.S. Pat. No. 4,994,559; U.S. Pat.No. 5,155,214; EP 470,183B; and Abraham et al., Quant. Biol. 51:657-668,1986) rat FGF-2 (see, Shimasaki et al., Biochem. Biophys. Res. Comm.,1988, and Kurokawa et al., Nucleic Acids Res. 16:5201, 1988), FGF-3,FGF-6, FGF-7 and FGF-9 are known (see also U.S. Pat. No. 5,155,214; U.S.Pat. No. 4,956,455; U.S. Pat. No. 5,026,839; U.S. Pat. No. 4,994,559, EP0,488,196 A2, EMBL or GenBank databases, and references discussedherein).

The effects of FGFs are mediated by high affinity receptor tyrosinekinases present on the cell surface of FGF-responsive cells (see, e.g.,PCT WO 91/00916, WO 90/05522, PCT WO 92/12948; Imamura et al., Biochem.Biophys. Res. Comm. 155:583-590, 1988; Huang et al., J. Biol. Chem.261:9568-9571, 1986; Partanen et al., EMBO J. 10:1347, 1991; andMoscatelli, J. Cell. Physiol. 131:123, 1987). Low affinity receptorsalso appear to play a role in mediating FGF activities. Cell typespecific expression of one or more of four FGF receptor genes that havebeen identified, plus additional receptor heterogeneity generated byalternative RNA splicing of the transcripts of such genes, may providethe basis for differential specificity of FGF family members amongdifferent tissues and cells.

For example, by way of illustration and not limitation, FGF-2 may besuitable for use in the present invention as a receptor binding ligandthat can be internalized by neuronal cells having surface FGF-2receptors. At physiologic concentrations FGF-2 may be trophic forinjured neurons, while at significantly lower concentrations FGF-2 isnot neurotrophic but may be readily internalized via neuronal FGF-2receptors. Accordingly, the use of sub-neurotrophic FGF-2 concentrationsin the present invention may provide a ligand that is not present insufficient quantities to create an FGF-2 sink, thereby avoiding theproblem of axonal entrapment associated with local administration ofneurotrophic factors, as discussed above. Those having skill in the artare familiar with routine methods for evaluating the local concentrationand bioavailability of FGF-2 provided as a ligand of the invention, forreadily detecting FGF-2 internalization by neurons and for determiningwhether a local FGF-2 sink sufficient to induce axonal entrapment hasaccumulated. (See, e.g., Logan et al., Prog. Growth Factor Res.5:379-405, 1994.)

(2) Neurotrophins

Neurotrophins (NT) comprise a multifunctional family of structurallyrelated proteins that may be useful as ligands in the present invention.NT regulate the developmental fates of neuronal cells during theformation and differentiation of neural networks, and provide essentialstimuli for the maintenance and survival of neural cells. NT may alsoregulate non-neuronal cells that express cell surface receptors specificfor one or more members of the NT family. The neurotrophin familyincludes nerve growth factor (NGF or NT-1), brain derived neurotrophicfactor (BDNF or NT-2), neurotrophin 3 (NT-3), neurotrophin 4 (NT-4/5),and neurotrophin 6 (NT-6). (See, e.g., Oshima et al., in Growth Factorsand Cytokines in Health and Disease, LeRoith and Bondy, eds., 229-258,1996 JAI Press, Greenwich, Conn.)

Nerve growth factor (NGF/NT-1), the prototype for the neurotrophinfamily, is a 26 kDa protein homodimer of 121 amino acid polypeptidesubunits, each derived from a 241 amino acid precursor. Mature NGFcontains three cysteine pairs involved in intrachain disulfide bondformation that is required for biological activity. NGF sequences arehighly conserved across species lines. Two distinct receptors for NGFare known. The high affinity NGF receptor trkA, encoded by the trk(tropomyosin receptor kinase) proto-oncogene, is a 140 kDa transmembraneglycoprotein that includes a cytoplasmic domain having tyrosine kinaseactivity. The trkA receptor is expressed on the surfaces of sensorycranial and dorsal root ganglia neurons, basal forebrain and caudateneurons, and on monocytes. The low affinity NGF receptor, which belongsto the distinct tumor necrosis factor receptor (TNF-R) superfamily, is a75 kDa transmembrane glycoprotein expressed by Schwann cells, neurons,lymphocytes, bone marrow fibroblasts, keratinocytes and myoepithelium,as well as on various tumor cell surfaces.

NGF exhibits a variety of biological activities within the NS includingthe CNS, including promotion of neuronal survival following axotomy,inhibition of apoptotic pathways and developmental regulation ofneuronal differentiation. (Hagg, in CNS Injuries: Responses andPharmacological Strategies (A. Logan and M. Berry, eds.) 1998 CRC Press,Boca Raton, Fla.; see also Oshima et al., in Growth Factors in Healthand Disease, LeRoith and Bondy, eds., 229-258, 1996 JAI Press,Greenwich, Conn.; Muller et al., J. Neurosci. Res. 38:41, 1994; Morimotoet al., Neuroreport 5:954, 1994, Tischler et al., J. Neurosci. 13:1533,1993; Oppenheim, Ann. Rev. Neurosci. 14:453, 1991; Hefti, J. Neruosci.6:2155, 1986; Oppenheim et al., J. Comp. Neurol. 210:174, 1982;Hamburger et al., J. Neurosci. 1:60, 1981.) NGF also appears to playsignificant roles in the regulation of hematopoiesis and inflammation,including reported modulation and/or stimulation of various lymphoid,myelomonocytic and granulocytic subpopulations.

Like NT-1, the other neurotrophins, brain derived neurotrophic factor(BDNF/NT-2), NT-3, NT-4/5 and NT-6, are 26 kDa homodimers that exhibit50-60% amino acid sequence homology with one another and that possessfunctional homology as well. (For a review of neurotrophic factors, seeHagg, in CNS Injuries: Responses and Pharmacological Strategies, A.Logan and M. Berry, eds., 1998 CRC Press, Boca Raton, Fla.) Allneurotrophins bind to the low affinity NGF receptor, while only NT-1 canbind trkA with high affinity. Related to the trkA receptor are theadditional neurotrophin receptors trkB, which binds NT-2 and NT-4/5, andtrkC, which binds NT-3. (Barbacid, in Growth Factors and Cytokines inHealth and Disease, LeRoith and Bondy, eds., 259-276, 1996 JAI Press,Greenwich, Conn.; Barbacid, J. Neurobiol. 25:1386, 1994; Squinto et al.,Cell 65:885, 1991; Lamballe et al., Cell 66:967, 1991; Klein et al.,Cell 61:647, 1990; Velenzuela et al., Neuron 10:963, 1990.)

Glial cell line-derived neurotrophic factor (GDNF) is a neurotrophicfactor that may be useful as a ligand in the present invention and thatis structurally unrelated to the NT family members. GDNF is a member ofthe transforming growth factor-β (TGF-β) gene superfamily and mayexhibit differential biological activity depending on the types ofneurons to which it is exposed. (See, e.g., McPherron et al., in GrowthFactors and Cytokines in Health and Disease, LeRoith and Bondy, eds.,357-393, 1996 JAI Press, Greenwich, Conn.)

(3) Antibodies and Other Ligands to Neuronal Cell Surface Molecules

As noted above, antibodies that specifically bind to neuronal cellsurface molecules may be useful as ligands in the present invention, andmay further include monoclonal or polyclonal antibodies, geneticallyengineered immunoglobulins or other natural, recombinant or syntheticproteins including chimeric fusion proteins that have antibody activity,or fragments of any of these immunoglobulins or immunoglobulinderivatives that specifically bind to neuronal cell surface molecules.Antibodies that are internalized by neuronal cells upon binding tocognate antigen on the neuronal cell surface as provided, as well asantibodies that may require an additional signal to be internalized,including but not limited to a signal that is the result of natural,genetically engineered or synthetic aggregation, crosslinking or inducedmultivalency, any of which may further include internalization that isinduced by the presence of multiple antibody binding sites havingspecificity for more than one cell surface antigenic determinant, arewithin the contemplated uses of antibodies as ligands in the presentinvention.

Genetically engineered antibodies that specifically bind to neuronalcell surface molecules may be useful as ligands in the presentinvention. For example, bacteriophage display selection methods may beuseful for producing single chain Fv immunoglobulins that demonstratehigh affinity binding to neuronal cell surface molecules. (see, e.g.,U.S. Pat. No. 5,223,409).

Neuronal cell surface molecules, to which antibodies that are to be usedas ligands in the invention as described above may specifically bind,may include any cell surface structure present on neurons that can beinternalized subsequent to ligand binding, including but not limited toproteins; glycoconjugates including glycoproteins, glycolipids,proteoglycans, glycosaminoglycans and the like; carbohydrates, lipids orother cell surface structures to which antibody may specifically bind.Markers for neuronal cell types, including neuronal cell surfacemarkers, are known in the art and may be readily determined by wellknown methodologies and reference literature, for example by way ofillustration and not limitation Lee et al. (Annu. Rev. Neurosci.19:187-217, 1996), Martini et al. (Glia 19:298-310, 1997), Rieger-Christet al. (Front. Biosci. 2:D348-D448, 1997) and Chao (Neuron 9:583-593,1992). Neuronal cell surface molecules may include, for example,neuronal cell adhesion molecule (NCAM), the polysialylatedoligosaccharide moiety of which has been reported to function as aninternalizable receptor for an antennapedia homeobox peptide. (Joliot etal., New Biol. (U.S.) 3:1121-1134, 1991) Neuronal cell surface moleculesmay also include, for example, the ganglioside GM₁, which has shown tofunction as receptor for cholera toxin B chain (see, e.g., Mulhein etal., J. Membr. Biol. 109:21, 1989); the proteoglycan syndecan, andvarious members of the integrin family of cell surface adhesionmolecules.

Other receptor-binding ligands may be used in the present invention. Anyprotein, polypeptide, analogue, or fragment that binds to a neuronalcell-surface receptor and is internalized may be used. These ligands maybe produced by recombinant or other means in preparation for conjugationto the nucleic acid binding domain. Ligands for use in the presentinvention may also be selected by a method such as phage display (see,e.g., U.S. Pat. No. 5,223,409) or variations of phage display with whichthose of ordinary skill in the art will be familiar, including methodsthat may be useful for selecting neuronal cell surface receptors havingparticularly low, particularly high or intermediate binding affinitiesfor neuronal cell surface receptors as those terms are understood bypersons of ordinary skill in the art with respect to certain knownneuronal cell surface receptors.

The DNA sequences and methods to obtain the sequences of thesereceptor-binding internalized ligands are well known. For example, theseligands and ligand/receptor pairs include urokinase/urokinase receptor(GenBank Accession Nos. X02760/X74309); α-1,3 fucosyl transferase,α1-antitrypsin/E-selectin (GenBank Accession Nos. M98825, D38257μM87862); P-selectin glycoprotein ligand, P-selectin ligand/P-selectin(GenBank Accession Nos. U25955, U02297/L23088), VCAM1 VLA-4 (GenBankAccession Nos. X53051/X16983); E9 antigen (Blann et al., Atherosclerosis120:221, 1996)/TGFβ receptor; Fibronectin (GenBank Accession No.X02761); type I α1-collagen (GenBank Accession No. Z74615), type Iβ2-collagen (GenBank Accession No. Z74616), hyaluronic acid/CD44(GenBank Accession No. M59040); CD40 ligand (GenBank Accession No.L07414)/CD40 (GenBank Accession No. M83312); ELF-3, LERTK-2 ligands(GenBank Accession Nos. L37361, U09304) for elk-1 (GenBank Accession No.M25269); VE-cadherin (GenBank Accession No. X79981); ligand forcatenins; ICAM-3 (GenBank Accession No. X69819) ligand for LFA-1, andvon Willebrand Factor (GenBank Accession No. X04385), fibrinogen andfibronectin (GenBank Accession No. X92461) ligands for α_(v)β₃ integrin(GenBank Accession Nos. U07375, L28832). DNA sequences of other suitablereceptor-binding internalized ligands may be obtained from GenBank orEMBL DNA databases, reverse-synthesized from protein sequence obtainedfrom PIR database or isolated by standard methods (Sambrook et al.,supra) from cDNA or genomic libraries.

b) Modification of Ligands

The ligands for use herein may be customized for a particularapplication. Briefly, additions, substitutions and deletions of aminoacids may be produced by any commonly employed recombinant DNA method.

Modification of the polypeptide may be effected by any means known tothose of skill in this art. The preferred methods herein rely onmodification of DNA encoding the polypeptide and expression of themodified DNA. DNA encoding one of the receptor-binding internalizedligands discussed above may be mutagenized using standard methodologies.For example, cysteine residues that may be useful to facilitateconjugation, such as formation of constructs or conjugates having adefined molar ratio of constituent polypeptides, can be added to apolypeptide. Conversely, cysteine residues that are responsible foraggregate formation may be deleted or replaced. If necessary, theidentity of cysteine residues that contribute to aggregate formation maybe determined empirically, by deleting and/or replacing a cysteineresidue and ascertaining whether the resulting protein aggregates insolutions containing physiologically acceptable buffers and salts. Inaddition, fragments of these receptor-binding internalized ligands maybe constructed and used. The binding regions of many of these ligands,for example that of FGF, have been delineated. The receptor bindingregion of FGF2 has been shown to reside between residues 33-77 andbetween 102-129 of the 155 amino acid form of FGF2, through the use ofFGF peptide agonists/antagonists and by mutation analysis. (Baird etal., PNAS 85:2324; Erickson et al., Biochem. 88:3441). Fragments ofligands may also be shown to bind and internalize by any one of thetests described herein. Modification of DNA encoding ligands may beperformed by a variety of methods, including site-specific orsite-directed mutagenesis of DNA encoding the protein and the use of DNAamplification methods using primers, as described above.

As noted above, binding to a receptor and subsequent internalization arethe only activities required for a ligand to be suitable for use herein.However, some of the ligands are growth factors and may have undesirablebiological activities, for example those that are mitogens. Althoughmature neurons may be generally regarded as non-dividing cells, andneuronal axon regeneration typically does not involve neuronal cellmitosis, ligands lacking mitogenic activity toward non-neuronal celltypes that may be present at sites of NS injury may be desirable in somesituations to avoid impairment of axonal regrowth that may result frommitogenic stimulation of such non-neuronal cells in the vicinity of aneuronal lesion. When present, the structural region of a ligandresponsible for inducing mitogenesis or any other such undesirablebiological activity may be altered in a manner that removes the unwantedactivity without ablating the ability to bind a receptor and beinternalized. Examples of suitable structural alteration of a ligand mayinclude, but need not be limited to, deletion of one or more nucleotidesfrom the appropriate region of a ligand-encoding DNA construct, mutationof nucleotides encoding one or more key amino acid residues upon whichthe unwanted biological activity depends, or genetically removing anentire domain encoding nucleotide sequence to remove the undesirableactivity and in its place substituting a functionally innocuous domainencoding sequence. For example, FGF muteins with reduced mitogenicactivity have been constructed by site-directed mutagenesis.

If the ligand has been modified so as to lack particular biologicalactivities, binding and internalization may still be readily assayed byany one of the following tests or other equivalent tests that areroutine and well known in the art. Generally, these tests involvelabeling the ligand, incubating it with target cells, and visualizing ormeasuring intracellular label. For example, briefly, the ligand may befluorescently labeled with fluorescein isothiocyanate (FITC), incubatedwith cells and examined by fluorescence microscopy or confocalmicroscopy for internalization. Alternatively, the ligand can beconjugated to a nucleic acid binding domain according to any of theconjugation methods described herein, complexed with a plasmid encodinga cytotoxic molecule and assessed for cytotoxicity after uptake byreceptor-bearing cells.

b. Other Elements that May be Included in a Construct

i. NABDs

As previously noted, nucleic acid binding domains (NABD) interact withthe DNA one is seeking to deliver in either a sequence-specific manneror a sequence-nonspecific manner. When the interaction is non-specific,the nucleic acid binding domain binds nucleic acid regardless of itssequence. For example, poly-L-lysine or poly-D-lysine is a basicpolypeptide that binds to oppositely charged DNA. Other highly basicproteins or polycationic compounds, including, but not limited to,histones, protamines, polyethylimine, spermine and spermidine, also bindto nucleic acids in a nonspecific manner. In addition, MnCl₂ and cobalthexamine also bind DNA and may serve to condense nucleic acid.

Many proteins have been identified that bind specific sequences of DNA.These proteins are responsible for genome replication, transcription andrepair of damaged DNA. The transcription factors regulate geneexpression and are a diverse group of proteins. These factors areespecially well suited for purposes of the subject invention because oftheir sequence-specific recognition. Host transcription factors havebeen grouped into seven well-established classes based upon thestructural motif used for recognition. The major families includehelix-turn-helix (HTH) proteins, homeodomains, zinc finger proteins,steroid receptors, leucine zipper proteins, the helix-loop-helix (HLH)proteins, and β-sheets. Other classes or subclasses may eventually bedelineated as more factors are discovered and defined. Proteins fromthose classes, or proteins that do not fit within one of these classesbut bind nucleic acid in a sequence-specific manner, such as SV40 Tantigen and p53, may also be used.

These families of transcription factors are generally well-known (seeGenBank; Pabo and Sauer, Ann. Rev. Biochem. 61:1053-1095, 1992; andreferences below). Many of these factors are cloned and the preciseDNA-binding region delineated in certain instances. When the sequence ofthe DNA-binding domain is known, a gene encoding it may be synthesizedif the region is short. Alternatively, the genes may be cloned from thehost genomic libraries or from cDNA libraries using oligonucleotides asprobes or from genomic DNA or cDNA by polymerase chain reaction methods.Such methods may be found in Sambrook et al., supra.

Helix-turn-helix proteins include the well studied λ Cro protein, λcI,and E. coli CAP proteins (see Steitz et al., Proc. Natl. Acad. Sci. USA79:3097-3100, 1982; Ohlendorf et al., J. Mol. Biol. 169:757-769, 1983).In addition, the lac repressor (Kaptein et al., J. Mol. Biol.182:179-182, 1985) and Trp repressor (Scheritz et al., Nature317:782-786, 1985) belong to this family. Members of the homeodomainfamily include the Drosophila protein Antennapaedia (Qian et al., Cell.59:573-580, 1989) and yeast MATα2 (Wolberger et al., Cell. 67:517-528,1991). Zinc finger proteins include TFIIIA (Miller et al., EMBO J.4:1609-1614, 1985), Sp-1, zif 268, and many others (see generally Krizeket al., J. Am. Chem. Soc. 113:4518-4523, 1991). Steroid receptorproteins include receptors for steroid hormones, retinoids, vitamin D,thyroid hormones, as well as other compounds. Specific examples includeretinoic acid, knirps, progesterone, androgen, glucocosteroid andestrogen receptor proteins. The leucine zipper family was defined by aheptad repeat of leucines over a region of 30 to 40 residues. Specificmembers of this family include C/EBP, c-fos, c-jun, GCN4, sis-A, andCREB (see generally O'Shea et al., Science 254:539-544, 1991). Thehelix-loop-helix (HLH) family of proteins appears to have somesimilarities to the leucine zipper family. Well-known members of thisfamily include myoD (Weintraub et al., Science 251:761-766, 1991);c-myc; and AP-2 (Williams and Tijan, Science 251:1067-1071, 1991). Theβ-sheet family uses an antiparallel β-sheet for DNA binding, rather thanthe more common α-helix. The family contains the MetJ (Phillips, Curr.Opin. Struc. Biol. 1:89-98, 1991), Arc (Breg et al., Nature 346:586-589,1990) and Mnt repressors. In addition, other motifs are used for DNAbinding, such as the cysteine-rich motif in yeast GAL4 repressor, andthe GATA factor. Viruses also contain gene products that bind specificsequences. One of the most-studied such viral genes is the rev gene fromHIV. The rev gene product binds a sequence called RRE (rev responsiveelement) found in the env gene. Other proteins or peptides that bind DNAmay be discovered on the basis of sequence similarity to the knownclasses or functionally by selection.

Several techniques may be used to select other nucleic acid bindingdomains (see U.S. Pat. No. 5,270,170; PCT Application WO 93/14108; andU.S. Pat. No. 5,223,409). One of these techniques is phage display.(See, for example, U.S. Pat. No. 5,223,409.) In this method, DNAsequences are inserted into gene III or gene VIII gene of a filamentousphage, such as M13. Several vectors with multicloning sites have beendeveloped (McLafferty et al., Gene 128:29-36, 1993; Scott and Smith,Science 249:386-390, 1990; Smith and Scott, Methods Enzymol.217:228-257, 1993). The inserted DNA sequences may be randomlygenerated, or may be variants of a known DNA-binding domain. Generally,the inserts encode from 6 to 20 amino acids. The peptide encoded by theinserted sequence is displayed on the surface of the bacteriophage.Bacteriophage expressing a desired nucleic acid-binding domain areselected for by binding to a preferred nucleic acid molecule fordelivery, for example, a neuronal therapeutic-encoding agent. Thistarget molecule may be single stranded or double stranded DNA or RNA.When the nucleic acid to be delivered is single-stranded, such as RNA,the appropriate target is single-stranded. When the molecule to bedelivered is double-stranded, the target molecule is preferablydouble-stranded. Preferably, the entire coding region of the nucleicacid molecule for delivery, such as a neuronal therapeutic agent, isused as the target. In addition, elements necessary for transcriptionthat are included for in vivo or in vitro delivery may be present in thetarget DNA molecule. Bacteriophage that bind the target are recoveredand propagated. Subsequent rounds of selection may be performed. Thefinal selected bacteriophage are propagated and the DNA sequence of theinsert is determined. Once the predicted amino acid sequence of thebinding peptide is known, sufficient peptide for use herein as annucleic acid binding domain may be made either by recombinant means orsynthetically. Recombinant means are used when the receptor-bindinginternalized ligand/nucleic acid binding domain is produced as a fusionprotein. In addition, the peptide may be generated as a tandem array oftwo or more peptides, in order to maximize affinity or binding ofmultiple DNA molecules to a single polypeptide.

ii. Promoters

In general, constructs will also contain elements necessary fortranscription and translation. In certain embodiments of the presentinvention, cell type preferred or cell type specific expression of aneuronal therapeutic-encoding gene may be achieved by placing the geneunder regulation of a promoter. The choice of the promoter will dependupon the cell type to be transformed and the degree or type of controldesired. Promoters can be constitutive or active and may further be celltype specific, tissue specific, individual cell specific, eventspecific, temporally specific or inducible. Cell-type specific promotersand event type specific promoters are preferred. Examples ofconstitutive or nonspecific promoters include the SV40 early promoter(U.S. Pat. No. 5,118,627), the SV40 late promoter (U.S. Pat. No.5,118,627), CMV early gene promoter (U.S. Pat. No. 5,168,062), andadenovirus promoter. In addition to viral promoters, cellular promotersare also amenable within the context of this invention. In particular,cellular promoters for the so-called housekeeping genes are useful.Viral promoters are preferred, because generally they are strongerpromoters than cellular promoters.

Tissue specific promoters are particularly useful for expression inneuronal cells. Promoters should be active in neuronal cells, andpreferably will be inactive or will have only very low activity in othercell types likely to be present in the vicinity of the NS lesion siteswhere the compositions and methods of the invention are to beadministered. By using one of this class of promoters, an extra marginof specificity can be attained.

Neuronal cell specific promoters are especially useful for targetingneuronal therapeutic agent-encoding genes. For treating traumatizedneurons in which axonal regeneration, neuronal survival andre-establishment of neural connections between perikaryons and distalneuronal targets are desired outcomes, the following promoters areespecially useful: GAP43 promoter (deGroen et al., 1995, J. Mol.Neurosci, 66: 109-119), FGF receptor promoter; neuron specific enolase(NSE) promoter (Forss-Pelter et al., 1986 J. Neurosci. Res. 16: 141-151;Sakimura et al., 1995 Brain Res. Mol. Br. Res. 28:19).

Other promoters that may not be regarded as neuronal cell specificpromoters but that may be useful promoters in certain embodimentsinclude tie promoter (WO 96/09381; Korhonen et al., Blood 86:1828, 1995;GenBank Accession No. X60954; GenBankAccession No. S89716); VCAM-1promoter (Tademarco et al., J. Biol. Chem. 267:16323, 1992; GenBankAccession No. M92431); alpha V-beta3 integrin promoter (VIIIa-Garcia etal., Blood 3:668, 1994; Donahue et al., BBA 1219:228, 1994); ICAM-3promoter, expressed in tumor endothelium (Patey et al., Am. J. Pathol.148:465, 1996; Fox et al., J. Path. 177:369, 1995; GenBank Accession No.S50015); CD44 promoter (Hofmann et al., Cancer Res. 53:1516, 1993;Maltzman et al., Mol. Cell. Biol. 16:2283, 1996; GenBank Accession No.HUMCD44B); CD40 promoter (Pammer et al., Am. J. Pathol. 148:1387, 1996;GenBank Accession No. HACD40L; GenBank Accession No. HSCD405FR); andnotch 4 promoter (Uyttendaele et al., Development 122:2251, 1996).

Inducible promoters may also be used. These promoters include MMTV LTR(PCT WO 91/13160), inducible by dexamethasone, metallothionein,inducible by heavy metals, and promoters with cAMP response elements,inducible by cAMP. By using an inducible promoter, the nucleic acid maybe delivered to a cell and will remain quiescent until the addition ofthe inducer. This allows further control on the timing of production ofthe gene product.

Event-type specific promoters are active or up-regulated only upon theoccurrence of an event, such as tumorigenicity or viral infection. TheHIV LTR is a well known example of an event-specific promoter. Thepromoter is inactive unless the tat gene product is present, whichoccurs upon viral infection. Some event-type promoters are alsotissue-specific.

Additionally, promoters that are coordinately regulated with aparticular cellular gene may be used. For example, promoters of genesthat are coordinately expressed when a particular FGF receptor gene isexpressed may be used. Then, the nucleic acid will be transcribed whenthe FGF receptor, such as FGFR1, is expressed, and not when FGFR2 isexpressed.

If the nucleic acid binding domain binds in a sequence specific manner,the construct must contain the sequence that binds to the nucleic acidbinding domain. As described below, the target nucleotide sequence maybe contained within the coding region of the neuronal therapeuticencoding agent, in which case, no additional sequence need beincorporated. Additionally, it may be desirable to have multiple copiesof target sequence. If the target sequence is coding sequence, theadditional copies must be located in non-coding regions of the neuronaltherapeutic-encoding agent. The target sequences of the nucleic acidbinding domains are typically generally known. If unknown, the targetsequence may be readily determined. Techniques are generally availablefor establishing the target sequence (e.g., see PCT Application WO92/05285 and U.S. Ser. No. 586,769).

In addition to the promoter, repressor sequences, negative regulators,or tissue-specific silencers may be inserted to reduce non-specificexpression of the neuronal therapeutic agent or prodrug. Multiplerepressor elements may be inserted in the promoter region. Repression oftranscription is independent on the orientation of repressor elements ordistance from the promoter. For examples of useful regulatory sequences,see, e.g., Dunaway et al., Mol Cell Biol 17: 182-9, 1997; Gdula et al.,Proc. Natl. Acad. Sci. USA 93:9378-83, 1996, Chan et al., J. Virol.70:5312-28, 1996; Scott and Geyer, EMBO. J. 14:6258-67, 1995; Kalos andFournier, Mol. Cell. Biol. 15:198-207, 1995; Chung et al., Cell 74:505-14, 1993; and Haecker et al., Mol. Endocrinology 9:1113-1126, 1995.

In preferred embodiments, elements that increase the expression of thedesired product are incorporated into the construct. Such elementsinclude internal ribosome binding sites (IRES; Wang and Siddiqui, Curr.Top. Microbiol. Immunol 203:99, 1995; Ehrenfeld and Semler, Curr. Top.Microbiol. Immunol 203:65, 1995; Rees et al., Biotechniques 20:102,1996; Sugimoto et al., Biotechnology 12:694, 1994). IRES increasetranslation efficiency. As well, other sequences may enhance expression.For some genes, sequences especially at the 5′ end inhibit transcriptionand/or translation. These sequences are usually palindromes that canform hairpin structures. Any such sequences in the nucleic acid to bedelivered are generally deleted. Expression levels of the transcript ortranslated product are assayed to confirm or ascertain which sequencesaffect expression. Transcript levels may be assayed by any known method,including Northern blot hybridization, RNase probe protection and thelike. Protein levels may be assayed by any known method, includingELISA, western blot, immunocytochemistry or other well known techniques.

Other elements may be incorporated into the construct. In preferredembodiments, the construct includes a transcription terminator sequence,including a polyadenylation sequence, splice donor and acceptor sites,and an enhancer. Other elements useful for expression and maintenance ofthe construct in mammalian cells or other eukaryotic cells may also beincorporated (e.g., origin of replication). Because the constructs areconveniently produced in bacterial cells, elements that are necessary orenhance propagation in bacteria are incorporated. Such elements includean origin of replication, selectable marker and the like (see discussionbelow).

An additional level of controlling the expression of nucleic acidsdelivered to cells using the complexes of the invention may be providedby simultaneously delivering two or more differentially regulatednucleic acid constructs. The use of such a multiple nucleic acidconstruct approach may permit greater specificity in obtainingexpression of the delivered genes only in appropriate cells, forexample, by delivering a first construct encoding a neuronal therapeuticagent under control of a first promoter and a second construct thatencodes a gene product capable of regulating the first promoter.

Alternatively, a multiple nucleic acid construct approach may permittemporal regulation of the expression of delivered nucleic acidsequences. As a non-limiting example, a first nucleic acid construct mayprovide a first neuronal therapeutic-encoding agent under regulation bya first promoter, such as an FGF-encoding nucleic acid regulated by aCMV promoter; a second nucleic acid construct may provide a secondneuronal therapeutic-encoding agent regulated by a second promoter, suchas a BDNF-encoding nucleic acid regulated by a GAP43 promoter. Withoutwishing to be bound by theory, regulated gene expression of thisconstruct pair delivered to neuronal cells following NS injury mayprovide constitutive FGF biosynthesis for the life of the firstconstruct to promote neuronal survival, and transient BDNF biosynthesisto promote axonal sprouting during the GAP43 induction phase. Thosefamiliar with the art will appreciate that multiple levels of regulatedgene expression may be achieved in a similar manner by selection ofsuitable regulatory sequences, including but not limited to promoters,enhancers and other well known gene regulatory elements.

Typically, the constructs are plasmid vectors. A preferred construct isa modified pNASS vector (Clontech, Palo Alto, Calif.), which has nucleicacid sequences encoding an ampicillin resistance gene, a polyadenylationsignal and a T7 promoter site. Other suitable mammalian expressionvectors are well known (see, e.g., Ausubel et al., 1995; Sambrook etal., supra; Invitrogen catalogue, San Diego, Calif.; Novagen, Madison,Wis.; Pharmacia catalogue, Uppsala, Sweden; and others).

iii. Nuclear Translocation Signal

As used herein, a “nuclear translocation or targeting sequence” (NTS) isa sequence of amino acids in a protein that assist or mediatetranslocation of the protein into a cell nucleus. Examples of NTSs areset forth in Table 1 below. Comparison with known NTSs, and if necessarytesting of candidate sequences, should permit those of skill in the artto readily identify other amino acid sequences that function as NTSs.The NTS may be derived from another polypeptide, or it may be derivedfrom another region in the same polypeptide. The NTS is typicallysynthesized as a DNA sequence encoding the NTS and insertedappropriately into either the ligand or NABD gene sequence.

TABLE 1 SEQ ID Source Sequence* NO. SV40 large TPro¹²⁶LysLysArgLysValGlu 1 Polyoma large T Pro²⁷⁹ProLysLysAlaArgGluVal 2Human c-Myc Pro¹²⁰AlaAlaLysArgValLysLeuAsp 3 Adenovirus E1ALys²⁸¹ArgProArgPro 4 Yeast mat α2 Lys³IleProIleLys 5 c-Erb-A A.Gly²²LysArgLysArgLysSer 6 B. Ser¹²⁷LysArgValAlaLysArgLysLeu 7 C.Ser¹⁸¹HisTrpLysGlnLysArgLysPhe 8 c-Myb Pro⁵²¹LeuLeuLysLysIleLysGln 9 p53Pro³¹⁶GlnProLysLysLysPro 10 NucleolinPro²⁷⁷GlyLysArgLysLysGluMetThrLysGlnLysGluValPro 11 HIV TatGly⁴⁸ArgLysLysArgArgGlnArgArgArgAlaPro 12 FGF-1 AsnTyrLysLysProLysLeu 13FGF-2 HisPheLysAspProLysArg 14 FGF-3 AlaProArgArgArgLysLeu 15 FGF-4IleLysArgLeuArgArg 16 FGF-5 GlyArgArg — FGF-6 IleLysArgGlnArgArg 17FGF-7 IleArgValArgArg 18 *Superscript indicates position in protein

In order to deliver a nucleic acid to the nucleus, a construct of thepresent invention may also include an NTS. If the construct is designedsuch that the receptor-binding internalized ligand and linked nucleicacid binding domain is cleaved or dissociated in the cytoplasm, then theNTS should be included in a portion of the complex that remains bound tothe nucleic acid, so that, upon internalization, the construct will betrafficked to the nucleus. Thus, the NTS is preferably included in thenucleic acid binding domain, but may additionally be included in theligand in targeted constructs. An NTS is preferred if the neuronaltherapeutic-encoding agent is DNA. If the neuronal therapeutic-encodingagent is mRNA, an NTS may be omitted. The nuclear translocation sequence(NTS) may be a heterologous sequence or a may be derived from theselected ligand. All presently identified members of the FGF family ofpeptides contain an NTS (see, e.g., International Application WO91/15229 and Table 2). A typical consensus NTS sequence contains anamino-terminal proline or glycine followed by at least three basicresidues in a array of seven to nine amino acids (see, e.g., Dang etal., J. Biol. Chem. 264:18019-18023, 1989; Dang et al., Mol. Cell. Biol.8:4049-4058, 1988, and Table 1).

iv. Cytoplasm Translocation Signal

A cytoplasm-translocation signal sequence is a sequence of amino acidsin a protein that causes retention of proteins in the lumen of theendoplasmic reticulum and/or translocates proteins to the cytosol. Asignal sequence in mammalian cells is KDEL (Lys-Asp-Glu-Leu) (SEQ ID NO.19) (Munro and Pelham, Cell 48:899-907, 1987). Some modifications ofthis sequence have been made without loss of activity. For example, thesequences RDEL (Arg-Asp-Glu-Leu) (SEQ ID NO. 20) and KEEL(Lys-Glu-Glu-Leu) (SEQ ID NO. 21) confer efficient or partial retention,respectively, in plants (Denecke et al., EMBO. J. 11:2345-2355, 1992).

A cytoplasm-translocation signal sequence may be included in either thereceptor-internalized binding ligand or the nucleic acid binding domain,or in both. If cleavable linkers are used to link the ligand with thenucleic acid binding domain, the cytoplasm-translocation signal ispreferably included in the nucleic acid binding domain, which will staybound to the neuronal therapeutic-encoding agent. Additionally, acytoplasmic-translocation signal sequence may be included in thereceptor-internalized binding ligand, as long as it does not interferewith receptor binding. Similarly, the signal sequence placed in thenucleic acid binding domain should not interfere with binding to theneuronal therapeutic-encoding agent.

c. Preparation of Constructs Including Therapeutic DNA and OtherElements

Within the context of this invention, specificity of delivery in a celltype specific manner may be achieved using a construct as disclosedherein. The choice of construct to use will depend upon the nature ofthe target cells.

The constructs may be tested in vitro and in vivo for the desiredeffect. Thus, for example, if the nucleic acid encodes a neurotrophin,neuronal cell survival, neurite extension or rescue from apoptosis maybe measured. Neurite extension and other assays of neurotrophin activityare known in the art (Berry et al., Neurocytology 1996). Any of a numberof well accepted assays for induction of apoptosis may be used. Theseinclude, but need not be limited to, detection of annexin binding toexteriorized phosphatidyl serine in the plasma membrane outer leaflet(e.g., Fadok et al., J. Immunol. 148:2207-2216, 1992), detection ofproteolytic cleavage of specific peptide substrates by apoptosisassociated proteases (e.g., Nagata, Cell 88:355, 1997), detection of DNAfragmentation (e.g., Kerr et al., Br. J. Canc. 26:239, 1972; Wyllie,Nature 284:555, 1980; Arends et al., Am. J. Pathol. 136:593, 1990), orother assays for induction of programmed cell death.

a) Preparation of Constructs Containing DNA

As noted above, nucleic acids and oligonucleotides for use as describedherein can be synthesized by any method known to those skilled in theart (see, e.g., Sosnowski et al, 1996 J. Biol. Chem. 271:33647; WO93/01286, U.S. application Ser. No. 07/723,454; U.S. Pat. No. 5,218,088;U.S. Pat. No. 5,175,269; U.S. Pat. No. 5,109,124; all of which arehereby incorporated by reference). Compositions and methods for thepreparation of particular DNA constructs are well known in the art, suchthat those having ordinary skill in the art can readily select a nucleicacid sequence for use as a neuronal therapeutic encoding agent in aconstruct of the invention and incorporate such a sequence into anappropriate construct for propagation and/or expression of the neuronaltherapeutic agent. See, e.g., Ausubel et al., Current Protocols inMolecular Biology, Greene Publishing, 1995; Sambrook et al., 1989. Forexample, DNA can be manipulated and amplified by PCR and by using thestandard techniques described in Molecular Cloning: A Laboratory Manual,2nd Edition, Maniatis et al., eds., Cold Spring Harbor, N.Y. (1989).Additional examples of methods for preparing DNA for use in compositionsare provided above.

i. Complex and Toroid Formation in Constructs which Include a Ligand

Where constructs are prepared that include a ligand as provided herein,the receptor-binding internalized ligand/nucleic acid binding domain isincubated with the neuronal therapeutic-encoding or prodrug-encodedagent, preferably a circular DNA molecule, to be delivered underconditions that allow binding of the nucleic acid binding domain to theagent. Conditions for preparing such complexes and for theircondensation into a toroidal shape are described in detail, for example,in Sosnowski et al, 1996 J. Biol. Chem. 271:33647, and inPCT/US95/07164, which are hereby incorporated by reference in theirentireties.

The ability of a construct to bind nucleic acid molecules, the amount ofcompaction achieved, binding of the construct to a receptor, and/orinternalization into a cell, may all be conveniently assessed viamethods available in the art. See, e.g., the assays described inpublished International Application No. WO 96/36362, for example.

d. Formulation and Administration of Pharmaceutical Compositions

i. Definitions and Indications

The conjugates and complexes provided herein are useful in the treatmentof various acute and chronic NS injury as may result following acute orchronic NS injury resulting from physical transection/trauma,contusion/compression or surgical lesion, vascular pharmacologic insultsincluding hemorrhagic or ischemic damage, or from neurodegenerative orother neurological diseases including those having genetic and/orautoimmune components. As used herein, “treatment” means any manner inwhich the symptoms of a condition, disorder or disease are amelioratedor otherwise beneficially altered. Treatment also encompasses anypharmaceutical use of the compositions herein. As used herein,“amelioration” of the symptoms of a particular disorder refers to anylessening, whether permanent or temporary, lasting or transient, thatcan be attributed to or associated with administration of thecomposition.

As noted above, the compositions of the present invention are used totreat NS injury. In acute or chronic NS injury resulting fromhemorrhagic, ischemic, hypoxic, or surgical lesion or other NS trauma,neural connections may be damaged or severed. Restoration or protectionof neural pathways through induction of neuronal survival or directedaxonal regrowth along projection tracts may be desirable, in order tomaintain or re-establish continuous retrograde flow of neurotrophicand/or neuronal therapeutic factors from the distal neuronal target tothe neuronal cell body (perikaryon). As such, the present inventionprovides nucleic acid delivery vehicles that may bind to cell surfacemolecules (receptors) via a ligand and internalize, thus delivering anucleic acid molecule. The invention also encompasses nucleic aciddelivery vehicles that are internalized by non-specific mechanisms,including but not limited to adsorptive endocytosis, fluid phaseendocytosis/pinocytosis, altered membrane permeability or gene activatedmatrices, or other mechanisms for nucleic acid delivery to cells. Theinvention further encompasses nucleic acid delivery using recombinantadenovirus or bacteriophage vectors. Genetically modified adenovirusesand bacteriophage exhibiting specifically targeted altered host celltropism have previously been disclosed in U.S. application Ser. No.09/039,060, filed Mar. 13, 1998, and U.S. application Ser. No.08/920,396, filed Aug. 29, 1997, respectively, which are inherentlyincorporated by reference in their entireties.

ii. Preparation of Pharmaceutical Agents

Pharmaceutical carriers or vehicles suitable for administration of theconjugates and complexes provided herein include any such carriers knownto those skilled in the art to be suitable for the particular mode ofadministration. In addition, the conjugates and complexes may beformulated as the sole pharmaceutically active ingredient in thecomposition or may be combined with other active ingredients.

The conjugates and complexes can be administered by any appropriateroute, for example, orally, parenterally, including intravenously,intradermally, subcutaneously, or topically, in liquid, semi-liquid orsolid form and are formulated in a manner suitable for each route ofadministration. Preferred modes of administration depend upon the lesionsite to be treated. The conjugates and complexes may be formulated intoa gene activated matrix (GAM), which is described in greater detailbelow. The conjugates and complexes may be administered by implantationinto the site of the body to be treated therapeutically.

The conjugates and complexes herein may be formulated intopharmaceutical compositions suitable for topical, local, intravenous andsystemic application. For the various uses herein, local administrationat or near a lesion site is preferred. Effective concentrations of oneor more of the conjugates and complexes are mixed with a suitablepharmaceutical carrier or vehicle. As used herein an “effective amount”of a compound for treating a particular lesion is an amount that issufficient to partially or fully maintain, restore, or in some mannerre-establish the neural connections whose loss may be associated withthe injury. Such amount may be administered as a single dosage or may beadministered according to a regimen whereby it is effective. Repeatedadministration may be required to achieve the desired degree of neuronalregeneration.

The concentrations or amounts of the conjugates and complexes that areeffective requires delivery of an amount, upon administration, thatrestores functional ability and/or prevents undesirable sequelae to NSinjury. Typically, the compositions are formulated for single dosageadministration. Therapeutically effective concentrations and amounts maybe determined empirically by testing the conjugates and complexes inknown in vitro and in vivo systems, such as those described here;dosages for humans or other animals may then be extrapolated therefrom.

The construct is included in the pharmaceutically acceptable carrier inan amount sufficient to exert a therapeutically useful effect in theabsence of undesirable side effects on the patient treated. Theconstructs may be delivered as pharmaceutically acceptable salts, estersor other derivatives of the constructs include any salts, esters orderivatives that may be readily prepared by those of skill in this artusing known methods for such derivatization and that produce compoundsthat may be administered to animals or humans without substantial toxiceffects. It is understood that number and degree of side effects dependsupon the condition for which the conjugates and complexes areadministered. For example, certain toxic and undesirable side effectsare tolerated when treating life-threatening illnesses, such as tumors,that would not be tolerated when treating disorders of lesserconsequence. The concentration of construct in the composition willdepend on absorption, inactivation and excretion rates thereof, thedosage schedule, and amount administered as well as other factors knownto those of skill in the art.

Preferably, the conjugate and complex are substantially pure. As usedherein, “substantially pure” means sufficiently homogeneous to appearfree of readily detectable impurities as determined by standard methodsof analysis, such as thin layer chromatography (TLC), gelelectrophoresis, high performance liquid chromatography (HPLC), used bythose of skill in the art to assess such purity, or sufficiently puresuch that further purification would not detectably alter the physicaland chemical properties, such as enzymatic and biological activities, ofthe substance. Methods for purification of the compounds to producesubstantially chemically pure compounds are known to those of skill inthe art. A substantially chemically pure compound may, however, be amixture of stereoisomers. In such instances, further purification mightincrease the specific activity of the compound.

Solutions, pastes or suspensions used for perineural, parenteral,intradermal, subcutaneous, or topical application can include any of thefollowing components: a sterile diluent, such as water for injection,saline solution, fixed oil, polyethylene glycol, glycerine, propyleneglycol or other synthetic solvent; antimicrobial agents, such as benzylalcohol and methyl parabens; antioxidants, such as ascorbic acid andsodium bisulfite; chelating agents, such as ethylenediaminetetraaceticacid (EDTA); buffers, such as acetates, citrates and phosphates; andagents for the adjustment of toxicity such as sodium chloride ordextrose. Parenteral preparations can be enclosed in ampules, disposablesyringes or multiple dose vials made of glass, plastic or other suitablematerial.

If administered intravenously, suitable carriers include physiologicalsaline or phosphate buffered saline (PBS), and solutions containingthickening and solubilizing agents, such as glucose, polyethyleneglycol, and polypropylene glycol and mixtures thereof. Liposomalsuspensions may also be suitable as pharmaceutically acceptablecarriers. These may be prepared according to methods known to thoseskilled in the art.

Upon mixing or addition of the construct(s) with the vehicle, theresulting mixture may be a solution, suspension, gel, paste, semisolid,dispersion, emulsion or the like. The form of the resulting mixturedepends upon a number of factors, including the intended mode ofadministration and the solubility of the construct in the selectedcarrier or vehicle. The effective concentration is sufficient forameliorating the symptoms of the disease, disorder or condition treatedand may be empirically determined based upon in vitro and/or in vivodata, such as the data from the rat ophthalmic or spinal cord model. Ifnecessary, pharmaceutically acceptable salts or other derivatives of theconjugates and complexes may be prepared.

The active materials can also be mixed with other active materials, thatdo not impair the desired action, or with materials that supplement thedesired action, including viscoelastic materials, such as hyaluronicacid, which is sold under the trademark HEALON (solution of a highmolecular weight (MW of about 3 millions) fraction of sodiumhyaluronate; manufactured by Pharmacia, Inc. see, e.g., U.S. Pat. Nos.5,292,362, 5,282,851, 5,273,056, 5,229,127, 4,517,295 and 4,328,803),VISCOAT (fluorine-containing (meth)acrylates, such as,1H,1H,2H,2H-hepta-decafluoro-decylmethacrylate; see, e.g., U.S. Pat.Nos. 5,278,126, 5,273,751 and 5,214,080; commercially available fromAlcon Surgical, Inc.), ORCOLON (see, e.g., U.S. Pat. No. 5,273,056;commercially available from Optical Radiation Corporation),methylcellulose, methyl hyaluronate, polyacrylamide andpolymethacrylamide (see, e.g., U.S. Pat. No. 5,273,751). Theviscoelastic materials are present generally in amounts ranging fromabout 0.5 to 5.0%, preferably 1 to 3% by weight of the constructmaterial and serve to coat and protect the treated tissues. Thecompositions may also include a dye, such as methylene blue or otherinert dye, so that the composition can be seen when injected into theeye or contacted with the surgical site during surgery.

The active materials can also be mixed with other active materials thatdo not impair the desired action, or with materials that supplement thedesired action, such as gene activated matrices described below, whichmay impair undesirable scar tissue formation.

Finally, the compounds may be packaged as articles of manufacturecontaining packaging material, one or more conjugates and complexes orcompositions as provided herein within the packaging material, and alabel that indicates the indication for which the construct is provided.

iii. Administration

Typically a therapeutically effective dosage should result from localapplication at NS lesion sites and should provide about 1 ng up to 100μg of active ingredient, preferably about 1 ng to about 10 μg per singledosage administration. It is understood that the amount to administerwill be a function of the construct selected, the indication treated,and possibly the side effects that will be tolerated.

Therapeutically effective concentrations and amounts may be determinedfor each application herein empirically by testing the conjugates andcomplexes in known in vitro and in vivo systems (e.g., murine, rat,rabbit, or baboon models), such as those described herein; dosages forhumans or other animals may then be extrapolated therefrom. The ratoptic nerve lesion model is a recognized model for studying the effectsof locally applied therapeutics and is described hereinbelow in theExamples.

The active ingredient may be administered at once, or may be dividedinto a number of smaller doses to be administered at intervals of time.It is understood that the precise dosage and duration of treatment is afunction of the disease being treated and may be determined empiricallyusing known testing protocols or by extrapolation from in vivo or invitro test data. It is to be noted that concentrations and dosage valuesmay also vary with the severity of the condition to be alleviated. It isto be further understood that for any particular subject, specificdosage regimens should be adjusted over time according to the individualneed and the professional judgment of the person administering orsupervising the administration of the compositions, and that theconcentration ranges set forth herein are exemplary only and are notintended to limit the scope or practice of the claimed compositions.

As provided, the present invention overcomes a problem associated withtherapies in the prior art that are directed to therapeutic delivery tothe CNS because the active ingredient does not have to traverse theblood brain barrier (BBB). It is well known in the art that the BBB actsas a selective molecular filter that may exclude compositions from theCNS. Accordingly, the compositions and methods provided herein for CNSdelivery of neuronal therapeutic agents, including neuronal therapeuticencoding agents, permit specific therapeutic uptake by CNS cells whileavoiding the limitations placed on delivered agents that must traversethe selective mechanisms of the BBB.

4. Methods of Promoting Neuronal Survival and Regeneration

Gene activated matrix (GAM) comprising nucleic acid encoding a neuronaltherapeutic agent may be administered to the vicinity of an injured ordiseased neuron, for example via a semi-solid gel comprising the geneactivated matrix that is inserted surgically at the injury site.Alternatively the GAM may be injected into the injury site as a liquidand then induced to form a gel, for example as a fibrin clot. Asdescribed above, a neuronal therapeutic encoding agent undergoes axonaldelivery of therapeutic DNAs via uptake and retrograde transport to thecell body of the neuron. The therapeutic DNA is delivered to the axonvia the gene activated matrix. The therapeutic DNA comprises an inactiveprodrug that is transcribed and translated within a neuronal cell toexpress an active neuronal therapeutic protein factor. The activeneurotropic protein factor stimulates axonal outgrowth into the geneactivated matrix, which in turn delivers more therapeutic DNA (prodrug)that is expressed as active neurotrophin. Upon activation of the growthresponse, neurons secrete matrix-degrading enzymes to facilitate axonalregrowth through the wound.

The delivery and expression of neuronal therapeutic encoding geneswithin a gene activated matrix to promote neuronal survival andregeneration may be assessed in any number of in vivo model systems. Inparticular, a lesioned rat optic nerve repair model or a regeneratingrat spinal cord model may be used. In each animal model, experimentallydamaged nerves are treated with a gene activated matrix that providestargeted delivery of a gene encoding a neuronal therapeutic agent or areporter gene. If the gene encodes a reporter, the reporter product isassayed post mortem. If the gene encodes a neuronal therapeutic agent,the neuronal therapeutic protein is assayed and regeneration of thedamaged nerve is analyzed post mortem. Moreover, any assayable geneproduct may be used. For reporter genes a wide variety of suitable genesare available. As described above, such reporters include but need notbe limited to β-galactosidase, alkaline phosphatase, β-glucuronidase,green fluorescent protein, large T antigen, and any protein for which anantibody exists or can be developed. Antibodies to the neuronaltherapeutic agent may be developed for immunohistochemical analysis orWestern blot analysis of regenerating neurons. Neuronal therapeuticagents are described herein.

The delivery and expression of neuronal therapeutic agent encoding geneswithin a gene activated matrix to promote neuronal survival andregeneration may be assessed in in vitro model systems. In particular,target cells are grown in culture and incubated with the gene activatedmatrix comprising a neuronal therapeutic agent-encoding gene or areporter gene. Moreover, any assayable gene product may be used. Thereporter gene product or the neuronal therapeutic encoding agent geneproduct may be assayed as described above.

The following examples are included for illustrative purposes only andare not intended to limit the scope of the invention.

EXAMPLES Example 1 Preparation of Gene Activated Matrix ContainingFGF2-Poly-L-Lysine Complexed with a Plasmid Encoding GFP Protein (GFP)Reporter Gene Under Promoter Regulation

Plasmid isolation, production of competent cells, transformation andmanipulations using the M13 cloning vectors are performed as described(Sambrook et al., Molecular Cloning, a Laboratory Manual, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., 1989). DNA fragmentsare purified using the Geneclean II kit, purchased from Bio 101 (LaJolla, Calif.). Recombinant DNA constructs are sequenced using theSequenase kit (version 2.0, United States Biochemical, Cleveland, Ohio)according to the manufacturer's instructions. Conjugation of FGF2 topoly-L-lysine K₈₄ homopolymer to produce FGF2-K is as described bySosnowski et al. (1996 J. Biol. Chem. 271:33647-33653). Preparation ofFGF2-poly-L-lysine complexed with a plasmid encoding green fluorescentprotein (GFP) under CMV promoter regulation is also essentially asdescribed above for FGF2-K complexed with a plasmid encodingβ-galactosidase above, except that a plasmid encoding GFP under CMVpromoter control (pEGFP, Clontech, Palo Alto, Calif.) was used insteadof the galactosidase construct.

Fibrin matrices to be used for the assembly of GAM are produced usingthe TISSEEL™ Kit (ImmunoAG, Vienna, Austria) according to themanufacturer's instructions. Briefly, lyophilized TISSEEL™ materialcontaining human fibrinogen, plasma fibronectin, factor XIII andplasminogen is reconstituted in a solution containing variousconcentrations of FGF2-K-GFP and bovine aprotinin following the TISSEEL™manufacturer's recommendations to form a first component that ismaintained at 37° C. for at least 10 min. Lyophilized human thrombinprovided in the kit is reconstituted with 40 mM CaCl₂ to form a secondcomponent, which is also held at 37° C. prior to use. Equal volumes ofthe first and second components are then mixed to initiate fibrinformation and drawn into capillary tubing (Accupette™, Dade Diagnostics,Inc., Aguada, Puerto Rico) to cure (usually 15-30 min at roomtemperature), after which the matrix is extruded sterilely and cut intosections for implantation at CNS lesion sites.

Collagen GAMs are prepared by lyophilizing FGF2-K or K condensatesprepared as described above but using DNA encoding FGF2 or GFP, and thenreconstituting theses lyophilized condensates with 2 mg sterile collagenpaste (Collagen Corporation, Palo Alto, Calif.) in sterile petri dishes.Alternatively, GAM plugs are formed by adding 3 ml of Cell Prime™collagen (1.5 mg/ml in DMEM, Collagen Corporation) to 1 ml of FGF2-K-DNA(50 μg DNA, 100 μg FGF2-K), pipeting 100 μl aliquots onto a dryice-chilled foil freezing substrate (prepared using aluminum foil formedinto dimples on an empty plastic 1000 μl pipette tip rack) andlyophilizing the plugs. Each plug (2.34 μg DNA, 4.68 μg FGF2-K, 46.8 μgcollagen) is rehydrated with one microliter of sterile water prior toits implantation at a CNS lesion site.

The following GAMs containing FGF-targeted GFP encoding plasmids areprepared according to these methods:

Matrix Targeting Reporter Gene GAM Component Agent Linker Encoding DNAK-GFP/ collagen none poly-L-lysine GFP collagen FGF2-K- collagen FGF2poly-L-lysine GFP GFP/ collagen K-GFP/fibrin fibrin none poly-L-lysineGFP FGF2-K- fibrin FGF2 poly-L-lysine GFP GFP/fibrin

Example 2 Preparation of DNA Construct Containing the Neuronal GAP43Promoter

Plasmid isolation, production of competent cells, transformation andmanipulations using the M13 cloning vectors are performed as described(Sambrook et al., Molecular Cloning, a Laboratory Manual, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., 1989). DNA fragmentsare purified using the Geneclean II kit, purchased from Bio 101 (LaJolla, Calif.). Recombinant DNA constructs are sequenced using theSequenase kit (version 2.0, United States Biochemical, Cleveland, Ohio)according to the manufacturer's instructions. DNA containing the humanGAP43 promoter sequence (Genbank accession number X840768) is obtainedas described in de Groen et al. (J. Mol. Neurosci. 6:109-119, 1995) andincorporated into plasmids in operative linkage with reporter geneencoding or neuronal therapeutic agent encoding sequences.

Example 3 Delivery and Expression of Targeted GFP Gene in Lesioned RatOptic Nerve Repair Model

In this example, targeted delivery of the GFP reporter gene to rat opticnerve neurons is conducted using a ligand as a molecular targeting agentin an in vivo model of neuronal regeneration.

Transgene expression of neuronal cells in vivo following experimentallyinduced axonal lesion is monitored in the rat optic nerve repair model.See, e.g., Logan et al., Meth. Neurosci. 21:3-19, 1994, which is herebyincorporated by reference in its entirety. Adult rats are anesthetizedby intraperitoneal injection of physiological saline solution containingketamine (40 mg/kg), acepromazine (1.2 mg/kg) and xylazine (8 mg/kg).The optic nerve is accessed intraorbitally by a dorsolateral approachand severed by transection using manual pressure applied with surgicalforceps. (Berry et al., J. Neurocytol. 25:147-170, 1996). Care is takento avoid damaging the central retinal artery or the optic nerve sheath.The conjugate having the following components (in 1-20 μL) is injectedunder pressure using a glass microsyringe at the optic nerve lesionsite:

FGF2-Kn-pCMV promoter-GFP encoding gene], wherein

FGF2 is the ligand protein as described in Sosnowski et al. (1996 J.Biol. Chem. 271:33647-33653).

Kn is the poly-L-lysine linker as described in Sosnowski et al. (supra)and having n=84;

and pCMV promoter-GFP encoding gene is the plasmid described in Example1 and containing the GFP gene under regulation of CMV promoter, andfurther wherein SPDP conjugation and plasmid complex formation are asdescribed in Sosnowski et al. (1996 J. Biol. Chem. 271:33647-33653). Asimilar construct containing the lacZ gene encoding beta-galactosidaseinstead of GFP was also prepared. (See Example 1.)

Following injection the lesion site is closed by standard surgicalprocedures. At 7 days post lesion, retinas are dissected and wholemounts observed under the fluorescent microscope for the detection ofGFP. For the detection of beta-galactosidase activity, retinas are fixedin 4% paraformaldehyde and then incubated with Xgal, a substrate forthis enzyme. FIG. 1 illustrates expression of the reporter genesbeta-galactosidase and GFP in retinal ganglion cells.

Example 4 Delivery and Expression of FGF Gene in Lesioned Rat OpticNerve Repair Model

In this example, GAM delivery of a gene encoding the neuronaltherapeutic agent human FGF2 to rat optic nerve neurons is conductedusing a collagen GAM in the lesioned rat optic nerve model of in vivoneuronal repair. GAMs having neuronal therapeutic encoding agent DNAcondensed on poly-L-lysine linkers are prepared using type I collagen(67 mg/ml, Collagen Corporation, Palo Alto, Calif.) essentially asdescribed above in Example 1; GAMs have either no targeting agent or theFGF2 ligand as a molecular targeting agent.

Experimental lesion of the optic nerve is performed as in Example 3except that animals are not sacrificed until day 30 and day 100post-lesion. For each GAM preparation, surgery and GAM implantation atthe site of injury are conducted on a group of 20 animals, each dividedinto four sets of five animals. Optic nerve injury and GAM implantationare performed on both eyes. FIG. 2 is a schematic diagram illustratingplacement of a GAM at a neuronal lesion site and retrograde axonaltransport of neuronal therapeutic encoding agent to the perikaryon.

At day 30 post-lesion, a first set of animals from each treatment groupis injected intravitreally with 5 μl of 20% (w/v) biotinylated dextranamine (BDA, Molecular Probes, Eugene, Oreg.), a qualitative anterogradetracer of axonal regeneration when administered intravitreally. A secondset of animals from each group is injected with 1 μl of 20% BDA in theoptic nerve at a point 2 mm distal to the lesion site, followingsurgical access of the site. Administration of BDA in this fashionpermits quantification of optic nerve axonal regeneration when labeledretinal ganglion cells (RGC) are counted post mortem. (Berry et al., J.Neurocytol. 25:147-170, 1996.)

Also at day 30 post-lesion, a third set of animals is sacrificed andretinas collected and snap frozen in liquid nitrogen. The dissectedretinas are detergent solubilized and affinity extracted withheparin-Sepharose to isolate expressed FGF molecules for quantificationby Western immunoblot analysis according to Coffin et al. (Mol. Biol.Cell 6:1861-1873, 1995), which is hereby incorporated by reference.Bound proteins are resolved by SDS-polyacrylamide gel electrophoresis,blot transferred to a polyvinyldiflouride membrane and detectedradioimmunochemically or by chemiluminescence according to well knownmethods. As shown in FIG. 3, human FGF2 expression is readily detectablein rat retinas recovered from animals to which were administered GAMshaving either no targeting agent (lane C) or the FGF2 ligand as amolecular targeting agent (lane T). Under the conditions employed, ratFGF was not detectable in rat brain (lane B) using an antibody thatpreferentially binds to human FGF2.

At day 10 and 100 post-lesion, a fourth set of animals is sacrificed,perfusion fixed, and optic nerves and retinas are dissected andprocessed for histochemistry and immunohistochemistry. Briefly, animalsare re-anesthetized and perfused through the left ventricle atatmospheric pressure with the descending aorta clamped and both externaljugular veins incised with physiological saline for 1 min followed by 4%paraformaldehyde in 0.1M phosphate buffer, pH 7.2, for 5 min. Afterperfusion, optic nerves are dissected, dehydrated through a gradedalcohol series, embedded in a low melting point polyester wax and storedat 4° C. (Logan et al., Meth. Neurosci. 21:3-19, 1994) Longitudinaloptic nerve microtome sections (7 μm thickness) are cut with a cooledchuck, floated onto a 1% gelatin solution on slides and air dried.

For immunohistochemical identification of specific cellular componentswithin lesions, sections are dewaxed, rehydrated and soaked 5 min inphosphate buffered saline. Immunohistochemical staining of optic nervesections is performed according to established techniques (Berry et al.,J. Neurocytol. 25:147-170, 1996) using appropriate dilutions ofcommercially available primary antibodies specific for the markerproteins listed below. Detection is with fluorophore (FITC or TRITC) orperoxidase conjugated secondary antibodies (Vector Laboratories, Inc.,Burlingame, Calif.) and 3,3′,4,4′-diaminobenzidine (Vector) as aperoxidase substrate, all according to the supplier's recommendations:

Primary antibodies are: rabbit polyclonal anti-GAP43 (1:5000, G. Wilkin,Imperial College, London), rabbit anti-bovine glial fibrillary acidicprotein (astrocytic marker) (1:1000, Sigma, St. Louis, Mo.), rabbitanti-rat fibronectin (1:100, Dakopatts, Ltd., Carpinteria, Calif.),rabbit anti-mouse sarcoma laminin (1:100 Sigma), rabbit anti-ratcarbonic anhydrase-II (oligodendrocyte marker) (1:5000, N. Gregson,UMDS, London), anti-rat monocyte marker ED1 (1:200, Serotec, Ltd.,Oxford, UK), anti-rat monocytic OX47 (Serotec), anti-RT97 (neurofilamentmarker) (1:200, Serotec), rabbit anti-tenascin, monoclonal mouseanti-chondroitin-6-sulphate proteoglycan.

Briefly, antibodies are diluted as indicated above in PBS containing 1%(w/v) bovine serum albumin, and 60 μl are applied to sections at 4° C.overnight. Slides are immersion washed twice in PBS, and then incubatedfor one additional hour in appropriate secondary antibodies (FITC- orTRITC-labeled anti-Ig or ABC kit for HRP detection, all from VectorLaboratories). Slides are washed twice in PBS and HRP labeled sectionsare developed with diaminobenzidine (Vector). Slides are then examinedand evaluated using immunofluorescence microscopy (FITC, TRITC) and darkground illumination microscopy (HRP) as described. (Berry et al., 1996).

For three-color analysis, the primary antibodies are polyclonalanti-glial fibrillary acidic protein antisera (1:1000, AdvancedImmunochemical, Inc., Long Beach, Calif.), mouse monoclonal IgGanti-GAP-43 (1:100, Sigma, St. Louis, Mo.) and affinity purifiedpolyclonal rabbit anti-laminin antibodies (1:100, Sigma). Sections arepretreated for 1 hr at room temperature with 60 μl 1.5% (v/v) normalgoat serum in 0.1% bovine serum albumin/PBS, then incubated overnight at4° C. in 60 μl of a cocktail containing the three primary antibodiesdiluted to the working concentrations indicated above in 0.1% BSA/PBS.The next day, slides are washed twice in PBS and incubated one hour inthe dark with FITC-goat anti-mouse IgG (Vector Laboratories, Burlingame,Calif.) according to the manufacturer's instructions, then washed twicein PBS again. Rhodamine conjugated goat anti-guinea pig Ig (Chemicon,Temecula, Calif.) is then applied to the sections for an hour accordingto the manufacturer's instructions, and the slides are again twicewashed prior to incubation for one hour in biotinylated goat anti-rabbitIgG (Vector Laboratories, Burlingame, Calif.) according to themanufacturer's instructions. Slides are then washed, incubated inavidin-AMCA conjugate (Vector Laboratories) according to the supplier'srecommendations, washed again and fixed in 2% paraformaldehyde prior tovisualization by immunofluorescence microscopy using a microscopeequipped with a UV light source and filter sets according to themanufacturers' specifications for each fluorophore.

At day 30 and 100 post-lesion, an additional set of animals from eachtreatment group is injected with 1 μl of 20% (w/v) rhodamine dextranamine (RDA, Molecular Probes, Eugene, Oreg.) in the proximal end of thetransected optic nerve following surgical access of the site.Administration of RDA in this fashion permits quantification of neuronalsurvival when labeled retinal ganglion cells (RGC) are counted postmortem. (Berry et al., J. Neurocytol. 25:147-170, 1996.) FIGS. 4 and 5depict neuronal survival 40 days after injury (FIG. 4) and 100 daysafter injury (FIG. 5) in animals to which GAMs were administered havingeither condensed neuronal therapeutic encoding agent DNA (GFP or humanFGF2) but no targeting agent (cGAM) or condensed neuronal therapeuticencoding agent DNA linked to the FGF2 ligand as a molecular targetingagent (tGAM).

The following GAMs containing FGF-targeted FGF encoding plasmids areprepared according to the methods of Example 1:

Reporter Molecular Gene Matrix Targeting Encoding GAM ComponentComponent Linker DNA FGF2-K- Type I collagen FGF2 poly-L-lysine GFP GFP/(K₈₄₋₁₀₀) collagen FGF2-K- Type I collagen FGF2 poly-L-lysine FGF2 FGF/(K₈₄₋₁₀₀) (human) collagen K-FGF/ Type I collagen none poly-L-lysineFGF2 collagen (K₈₄₋₁₀₀) (human) K-GFP/ Type I collagen nonepoly-L-lysine GFP collagen (K₈₄₋₁₀₀)

Example 5 Preparation of a Cholera Toxin B-Chain Targeted Conjugate forDelivery of a Neuronal Therapeutic Encoding Agent

A. Derivatization of Poly-L-lysine (K₁₀₀ with SPDP.

Poly-L-lysine (K₁₀₀) is modified with a 1.5 molar excess ofN-succinimidyl-3-[2-pyridyldithio]proprionate (SPDP, Pierce ChemicalCo., Rockford, Ill.) for 30 min at room temperature in conjugationbuffer (0.1 M PO₄-pH 8.0, 0.1 M NaCl, 1 mM EDTA) and unreacted SPDP isremoved by diafiltration using a 10 kDa cutoff membrane. PDPconcentration is determined by optical density at 314 nm and K₈₂concentration is determined using the BCA protein assay. PDP-K₈₂ isreduced for 10 min at room temperature by the addition of dithiothreitol(DTT) to a final concentration of 5 mM to yield sulfhydryl modifiedK₁₀₀. Excess DTT is removed by diafiltration.

B. Derivatization of CTb with SPDP Cholera Toxin B Chain (CTb,Calbiochem, San Diego, Calif.) is equilibrated in conjugation buffer andreacted with a 5-fold molar excess of SPDP for 30 min at roomtemperature, after which unreacted SPDP is removed by diafiltration. PDPand CTb concentrations are measured as described above to determine aPDP:CTb molar ratio of 2-3.C. Conjugation of SH-K₁₀₀ to CTb-PDP and Purification of ConjugateSH-K₁₀₀ and CTb-PDP are combined at a molar ratio of 1:1.5 and reactedovernight at 4° C. The reaction is terminated by removal of unreactedCTb-PDP using a Resource S™ column (Pharmacia, Inc., Piscataway, N.J.)equilibrated in buffer A (0.1 M PO₄-pH 8.0, 1 mM EDTA) and eluted withtwo column volumes of the same buffer followed by a step gradient ofthree column volumes of 10% buffer B (buffer A made 3M in NaCl) inbuffer A, then a 10%-70% buffer B gradient over 24 column volumes andthen four column volumes of buffer B undiluted. Pooled fractions in the20-40% B portion of the gradient contain CTb-K₁₀₀ conjugate and K₁₀₀,the latter being removed either by gel filtration chromatography using aSephacryl™ S200 column (Pharmacia) isocratically eluted with 10 mMHepes-pH 7.3-0.13 M NaCl, or by Butyl-650M (TosoHaas, Linton, UK)hydrophobic interaction chromatography of pooled Resources™ fractionsmade 1.5 M in ammonium sulfate.

Yield and purification of the conjugate are determined using absorbanceat 280 nm and BCA assay for protein quantification and integrationanalysis of chromatography peaks, plus LLS-particle size analysis. From5 mg of CTb starting material, 3 mg of final product is obtained.Biological activity of the conjugate is also determined, usingtransfection assays according to references cited herein. (See, e.g.,Sosnowski et al., 1996 J. Biol. Chem. 271:33647-33653.)

In order to test the target specificity of CTb conjugates, PC12 (ratpheochromocytoma) and BHK cells are plated on a 24 well plate andincubated for 48 hr with CTb-K-DNA_(GFP). GFP expression is analyzedunder an inverted fluorescent microscope. Representative results of suchanalysis are shown in FIG. 6.

Example 6 Neuronal Delivery and Expression of LacZ Gene in Lesioned RatSpinal Cord

In this Example, a rat model system is presented for introducingexperimental CNS lesions and using FGF2 targeted condensed DNAs todeliver genes to injured neurons in the spinal cord. The first orderascending sensory system of the gracile tract and the descendingcorticospinal system are used to model spinal cord. Both tracts arefound in the dorsal funiculi and at the level of T8 are easily lesionedsurgically by contusion or section without disturbing the L4/5 rootentry zone.

The dorsal funiculus of the spinal cord is crushed at the level of T8 byforceps as follows: The surgical approach is standard through a partiallaminectomy, dura and arachnoid are incised, and the points of forcepsseparated to the medial margins of the dorsal root entry zone along thedorsolateral sulcus, and lowered to a depth of 2 mm. Approximation ofthe tips crushes the dorsal columns, including all the axons in theascending gracile tracts and the descending corticospinal tractsbilaterally. The pia remains intact and the patency of the overlyingvessels is preserved.

Targeted condensed DNA encoding the LacZ gene is prepared according toSosnowski et al. (1996 J. Biol. Chem. 271:33647-33653). Access to thelesion site for injection of the DNA is through the exposed piaoverlying the site of spinal cord transection. At 7 days post-lesion,animals are sacrificed and perfusion fixed as described in Example 3.Brains, spinal cords and dorsal root ganglia (DRG) are dissected andprocessed for beta-galactosidase histochemistry, also as described inExample 3. FIG. 7 illustrates representative fields showingbidirectional retrograde transfection as evidenced by beta-galactosidase(LacZ) gene expression.

Example 7 Delivery and Expression of Neuronal Therapeutic EncodingAgents in Regenerating Rat Spinal Cord

In this Example, a rat model system is presented for introducingexperimental CNS lesions and for using GAMs to deliver neuronaltherapeutic agent encoding genes to regenerating axons in the spinalcord. The first order ascending sensory system of the gracile tract andthe descending corticospinal system are used to model spinal cordregeneration using both short (15 and 30 days post lesion, dpl) and long(60 and 90 dpl) sampling times. Both tracts are found in the dorsalfuniculi and at the level of T8 are easily lesioned surgically bycontusion or section without disturbing the L4/5 root entry zone.

The dorsal funiculus of the spinal cord is crushed at the level of T8 byforceps as follows: The surgical approach is standard through a partiallaminectomy, dura and arachnoid are incised, and the points of forcepsseparated to the medial margins of the dorsal root entry zone along thedorsolateral sulcus, and lowered to a depth of 2 mm. Approximation ofthe tips crushes the dorsal columns including all the axons in theascending gracile tracts and the descending corticospinal tractsbilaterally. Both tracts are found in the dorsal funiculi and at thelevel of T8 are easily lesioned without disturbing the L4/5 root entryzone. The pia remains intact and the patency of the overlying vessels ispreserved.

GAMs are prepared according to Examples 1 and 4. Access to the lesionsite for implantation of the GAMs is through the exposed pia overlyingthe site of spinal cord transection. At intervals of 15, 30, 60 and 90days post-lesion, animals are sacrificed and perfusion fixed asdescribed in Example 4. Brains, spinal cords and dorsal root ganglia(DRG) are dissected and processed for histochemistry, also as describedin Example 4.

Neuronal regeneration and tissue scarring in the spinal cord lesion aremonitored as follows: Ipsilateral L4/5 DRG and pyramidal neurons inlayers V and VI of the ipsilateral sensorimotor cortex are retrogradelylabeled by injecting 2 μl of a 20% tracer solution (e.g., BDA, FDA) intothe cord lesion site 2 days prior to sacrifice. The regenerativeresponse of the gracile tract axons to injury is monitored qualitativelyby a lysinated rhodamine dextran amine (LRDA) transganglionic labelingtechnique after sciatic nerve injection, and that of the corticospinalaxons by labeling the pyramids on the ventral surface of the medullaoblongata. Axonal regeneration is detected by the presence of labeledaxons crossing the lesion and invading the distal tracts in serialsections through the lesion.

The number of ascending axons regenerating through the lesion isdetermined as follows: Regenerated gracile tracts are retrogradelylabeled following injection of 2 μl of 20% HRP (Sigma) into the lesion24 hr prior to autopsy. HRP is injected at Ti (7 segments rostral to thelesion site); the number of retrogradely HRP-filled ipsilateral L4/5dorsal root ganglia after this injection is scored by counting filledcells in serial sections through the ganglia. A quantitative measure ofcorticospinal tract regeneration is achieved by counting the numbers ofHRP filled pyramidal cells in layers V and VI of the ipsilateral andcontralateral sensorimotor neocortex after uptake at T13 (5 segmentscaudal to the lesion).

The above axon labeling methods are also used to examine re-innervationof targets both at the electron and light microscopic levels. In thesestudies HRP methods unequivocally identify regenerated DRG terminals inthe ipsilateral gracile nucleus, and corticospinal terminations on motorhorn cells below the lesion. Immunohistochemical analysis is essentiallyas described above in Example 4.

Example 8 Delivery and Expression of Neuronal Therapeutic EncodingAgents in Regenerating Rat Spinal Cord

In this Example, a rat model system is presented for introducingexperimental CNS lesions and using GAMs to deliver neuronal therapeuticencoding agents to modify scar deposition at the site of injury in thespinal cord model. The first order ascending sensory system of thegracile tract and the descending corticospinal system are used to modelspinal cord regeneration using both short (2 weeks post-lesion) and long(10 weeks post-lesion) sampling times. Both tracts are found in thedorsal funiculi and at the level of T8 are easily lesioned surgically bycontusion or section without disturbing the L4/5 root entry zone.

The injury is performed as described in Example 7.

GAMs are prepared according to Examples 1 and 4. Access to the lesionsite for implantation of the GAMs is through the exposed pia overlyingthe site of spinal cord transection. At intervals of 2 and 10 weekspost-lesion, animals are sacrificed and perfusion fixed as described inExample 4. Brains, spinal cords and dorsal root ganglia (DRG) aredissected and processed for immunohistochemistry, also as described inExample 4.

Evaluation of scar and injury tissue is based on the presence and sizeof cystic cavitations at the lesion epicenter. Surviving axons aredemonstrated using an anti-neurofilament antibody (i.e. RT97) asdescribed in Example 4. To evaluate the number of dividing cells (i.e.proliferating oligodendrocytes) rats receive daily injections of BrdU(Sigma, 50 mg/Kg, i.p.) for 7 days beginning at day 21 after injury. Fordetection of BrdU, tissue sections are treated with 2N HCl for 1 hr.,rinsed and then stained with an antibody against BrdU (Dako,Carpinteria, Calif.), using the protocol described in Example 4.Evaluation of the degree of myelination is performed by staining tissuesections with an antibody against myelin basic protein (MBP) using theprotocol described in Example 4.

Example 9 Delivery and Expression of Neuronal Therapeutic EncodingAgents in Regenerating Rat Spinal Cord Using Mixed GAM

In this Example, a rat model system is presented for introducingexperimental CNS lesions and using a GAM to deliver neuronal therapeuticencoding genes to regenerating axons in the spinal cord. The GAM in thisexample also contains live cells (i.e., fibroblasts) and for this reasonis called a mixed GAM. The GAMs are prepared, essentially as describedin Example 1 but in addition, the GAMs are supplemented with mammalian,preferably autologous cells. These cells serve to modify healing time,synthesize matrix and may internalize DNA present in the GAM. The firstorder ascending sensory system of the gracile tract and the descendingcorticospinal system are used to model spinal cord regeneration usingboth short (2 weeks post-lesion) and long (10 weeks post-lesion)sampling times. Both tracts are found in the dorsal funiculi and at thelevel of T8 are easily lesioned surgically by contusion or sectionwithout disturbing the L4/5 root entry zone.

The injury is performed as described in example 7.

GAMs are prepared according to Examples 1 and 4 with the modificationsdescribed above. Access to the lesion site for implantation of the GAMsis through the exposed pia overlying the site of spinal cordtransection. At intervals of 2 and 10 weeks post-lesion, animals aresacrificed and perfusion fixed as described in Example 4. Brains, spinalcords and dorsal root ganglia (DRG) are dissected and processed forimmunohistochemistry, also as described in Example 4.

Evaluation of scar tissue, cell proliferation, axonal growth andmyelination is performed as described in Example 8.

From the foregoing it will be appreciated that, although specificembodiments of the invention have been described herein for purposes ofillustration, various modifications may be made without deviating fromthe spirit and scope of the invention. Accordingly, the invention is notlimited except as by the appended claims.

1. A device for promoting neuronal survival or regeneration, comprising:a gene activated matrix comprising a biocompatible matrix and at leastone neuronal therapeutic encoding agent having an operably linkedpromoter. 2.-5. (canceled)
 6. The device of claim 1 wherein the promoteris a neuronal cell specific promoter.
 7. The device of claim 6 whereinthe promoter is selected from the group consisting of GAP43 promoter,FGF receptor promoter and neuron specific enolase promoter.
 8. Thedevice of claim 1 wherein the neuronal therapeutic encoding agentencodes a neurotrophic factor. 9.-11. (canceled)
 12. The device of claim1 wherein the neuronal therapeutic encoding agent encodes an inhibitorof an antagonist of axonal generation or regeneration. 13.-19.(canceled)
 20. The device of claim 1 wherein the neuronal therapeuticencoding agent is non-covalently associated with the gene activatedmatrix.
 21. The device of claim 1 wherein the neuronal therapeuticencoding agent is absorbed in or adsorbed to the gene activated matrix.22.-23. (canceled)
 24. A device for promoting neuronal survival orregeneration, comprising: a gene activated matrix; at least one supportcell; and at least one neuronal therapeutic encoding agent having anoperably linked promoter.
 25. (canceled)
 26. The device of claim 24wherein the support cell is selected from the group consisting of: aSchwann cell an oligodendrocyte an astrocyte, a microglial cell and afibroblast. 27.-31. (canceled)
 32. The device of claim 24 wherein thesupport cell is an inflammatory cell selected from the group consistingof a macrophage, a neutrophil, a monocyte, a granulocyte and alymphocyte.
 33. (canceled)
 34. The device of claims 1 or 24 wherein thegene activated matrix is an implant for a neuronal injury site. 35.-36.(canceled)
 37. The device of claims 1 or 24 wherein the gene activatedmatrix is a composition selected from the group consisting of asolution, a paste, a suspension, a powder, a semisolid, an emulsion anda gel. 38.-39. (canceled)
 40. The device of claims 1 or 24, furthercomprising a targeting agent, wherein said targeting agent is complexedwith or conjugated to the neuronal therapeutic encoding agent and iscapable of binding a neuronal cell surface receptor a repair cellsurface receptor, or extracellular matrix. 41.-45. (canceled)
 46. Thedevice of claims 1 or 24, further comprising a nucleic acid bindingdomain, wherein said nucleic acid binding domain binds to a nucleic acidsequence that forms a portion of the neuronal therapeutic encodingagent.
 47. The device of claims 1 or 24, further comprising at least onelinker that is selected from the group consisting of a cleavable linker,a linker that provides an intracellular protein sorting peptidesequence, a linker that reduces steric hindrance, a linker that providesa nuclear translocation signal and a linker that possesses a nucleicacid condensing ability.
 48. The device of any one of claims 1 or 24wherein the device contains sub-physiologic amounts of a neuronaltherapeutic agent.
 49. (canceled)
 50. A device according to claims 1 or24 or 25, further comprising a conduit having a lumen. 51.-58.(canceled)
 59. A method for transferring a neuronal therapeutic encodingagent into a neuronal cell, comprising: contacting a neuronal cell withthe device of claims 1 or 24 to effectively transfer the neuronaltherapeutic encoding agent into the neuronal cell. 60.-61. (canceled)62. The method of claim 59 wherein the device is contacted with aneuronal cell at a neuronal injury site. 63.-67. (canceled)
 68. A methodfor transferring a neuronal therapeutic encoding agent into a repaircell, comprising: contacting a repair cell with the device of claims 1or 24 to effectively transfer the neuronal therapeutic encoding agentinto the repair cell.
 69. The method of claim 68 wherein the device iscontacted with a repair cell at a neuronal injury site. 70.-76.(canceled)
 77. A method of preparing a gene activated matrix forpromoting neuronal regeneration and survival, comprising contacting aneuronal therapeutic encoding agent with a biocompatible matrix suchthat the neuronal therapeutic encoding agent associates non-covalentlywith the matrix. 78.-80. (canceled)